Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The internal transcribed spacer 2 (ITS2) of the eukaryotic ribosomal DNA (rDNA) cluster plays an essential role in processing of the ribosomal RNA, which is primarily accomplished by the secondary structures acquired by the molecule after transcription. Two possible structural conformation models have been proposed for the ITS2 region, the 'ring model' and the 'hairpin model,' and the former has been widely used in many molecular phylogenetic analyses incorporating structural information available to date. To evaluate the validity of this model, in vitro transcribed ITS2 molecules from species representing the three superfamilies of the Calyptratae clade (Diptera: Schizophora), namely Cochliomyia hominivorax, Musca domestica, and Glossina morsitans, were submitted to enzymatic digestion with single- and double-stranded specific nucleases (RNases I, A, T1, and V1). The resulting fragments were analyzed by capillary electrophoresis and digestion sites were mapped in the secondary structure models which were obtained by in silico prediction with further refinement by homology comparisons. The pattern of RNA fragments generated by these RNases show a high degree of correlation to most of the predicted helix-loop regions and structural motifs. Discrepancies to the models can be explained by alternative structural conformation dynamics (in M. domestica and G. morsitans) and by higher-order factors (such as tertiary interactions) that may stabilize thermodynamically unfavored structures (in C. hominivorax).763158171Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/61217-3]