This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A + T-rich sequence is linked to three features: a high A + T content throughout the entire gene, a high A + T content in the third codon position, and a predominance of A + T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species. © 2005 Taylor & Francis Ltd.162156160Beard, C.B., Hamm, D.M., Collins, F.H., The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects (1993) Insect Mol Biol, 2, pp. 103-124Bernasconi, M.V., Valsangiacomo, C., Piffaretti, J.C., Ward, P.I., Phylogenetic relationships among Muscoidea (Diptera: Calyptratae) based on mitochondrial DNA sequences (2000) Insect Mol Biol, 9, pp. 67-74Caterino, M.S., Cho, S., Sperling, F.A.H., The current state of insect molecular systematics: A thriving Tower of Babel (2000) Annu Rev Entomol, 45, pp. 1-54Crozier, R.H., Crozier, Y.C., The mitochondrial genome of the honeybee Apis mellifera: Complete sequence and genome organization (1993) Genetics, 133, pp. 97-117Foster, P.G., Jermiin, L.S., Hickey, D.A., Nucleotide composition bias affects amino acid content in proteins coded by animal mitochondria (1997) J Mol E, 44, pp. 282-288Greenberg, B., Flies and disease (1973) Biology and Disease Transmission, 2. , New Jersey: University Press PrincetownInfante, M.E., Azeredo-Espin, A.M.L., Genetic variability in mitochondrial DNA of screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil (1995) Biochem Genet, 33, pp. 737-756Kumar, S., Tamura, K., Nei, M., (1993) MEGA: Molecular Evolutionary Genetics Analysis, Version 1.01, , University Park, Pennsylvania: The Pennsylvania State UniversityLessinger, A.C., Azeredo-Espin, A.M.L., Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing flies (2000) Med Vet Entomol, 14, pp. 71-80Litjens, P., Lessinger, A.C., Azeredo-Espin, A.M.L., Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA (2001) Med Vet Entomol, 15, pp. 183-188Lunt, D.H., Zhang, D.-X., Szymura, J.M., Hewitt, G.M., The insect cytochrome oxidase I gene: Evolutionary patterns and conserved primers for phylogenetic studies (1996) Insect Mol Biol, 5, pp. 153-165Morlais, I., Severson, D.W., Complete mitochondrial DNA sequence and amino acid analysis of the cytochrome c oxidase subunit I (COI) from Aedes aegypti (2002) DNA Seq, 13, pp. 123-127Nirmala, X., Hypsa, V., Zurovec, M., Molecular phylogeny of Calyptratae (Diptera: Brachycera): The evolution of 18S and 16S ribosomal rDNAs in higher dipterans and their use in phylogenetic inference (2001) Insect Mol Biol, 10, pp. 475-485Saccone, C., De Giorgi, C., Gissi, C., Pesole, G., Reyes, A., Evolutionary genomics in Metazoa: The mitochondrial DNA as a model system (1999) Gene, 238, pp. 195-209Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers (1994) Ann Entomol Soc Am, 87, pp. 651-701Szalanski, A.L., Owens, C.B., Sequence change and phylogenetic signal in muscoid COII DNA sequences (2003) DNA Seq, 14, pp. 331-334Tajima, F., Nei, M., Estimation of evolutionary distance between nucleotide sequences (1984) Mol Biol Evol, 1, pp. 269-285Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools (1997) Nucleic Acids Research, 25, pp. 4876-4882