Kefir is a fermented milk elaborated with kefir grains. It constitutes an ecological system composed by a protein-polysaccharide matrix on which a complex microbiota is naturally immobilized. The objective of this work was to develop an antibody-based method in order to achieve a simple and rapid way to quantify Lactobacillus kefir in a complex environment. Rabbit antisera against isolated S-layer proteins from L. kefir and L. parakefir were obtained. Both antisera recognized the 21 strains of L. kefir and the three strains of L. parakefir analysed by dot-blot. By indirect ELISA, specific antiserum against L. kefir S-layer showed reactivity against pure cultures of both L. kefir and L. parakefir, but did not react with L. brevis cells. Competitive ELISA allowed quantification of L. kefir at concentrations ranging from 5 × 10 5 to 108 bacteria ml-1, but neither L. parakefir nor L. brevis pure cultures produced inhibition. Through this approach it was possible to detect 108 L. kefir ml-1 in samples of milk fermented for 48 h at 20°C with different kefir grains. Competitive ELISA developed in our laboratory is a suitable method to detect and quantify L. kefir in milk and in consequence it could be applied to analyse the relationship between the composition of microflora and probiotic and technological characteristics of the products.Centro de Investigación y Desarrollo en Criotecnología de AlimentosFacultad de Ciencias Exactas