Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates ofLact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies usingEco RI and HindIII as restriction enzymes, and cDNA fromEscherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, EcoRI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When HindIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when EcoRI and HindIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake.