Screening of sourdough lactic acid bacteria for bacteriocin production resulted in the isolation of a Gram-positive, catalase-negative, non-spore-forming, non-motile rod bacterium (strain CS1(T)) that could not be associated with any previously described species. Comparative 16S rRNA gene sequence analysis recognized strain CS1(T) as a distinct member of the genus Lactobacillus. By a species-specific PCR strategy, five additional strains previously isolated from sourdoughs T were found to belong to the same species as strain CS1(T), as confirmed by 16S rRNA gene sequence analysis. The closest related species were Lactobacillus durianis, Lactobacillus malefermentans and Lactobacillus suebicus, with which strain CS1(T) shared 93% sequence similarity. For a further characterization of strain CS1(T), physiological (growth temperature, CO2 production, hydrolysis of arginine, isomeric type of lactate, sugar fermentation) and chemotaxonomic (G + C content and peptidoglycan structure) properties were determined. Phenotypic characterization showed that strain CS1(T) was a member of the obligately heterofermentative group of the genus Lactobacillus. The DNA G + C content was 44(.)6 mol%. The peptidoglycan was of the A3alpha (L-LyS-L-Ser-L-Ala(2)) type. Physiological, biochemical and genotypic data, as well as results of DNA-DNA hybridization of genomic DNA with one of the closest phylogenetic relatives, L. durianis (34(.)3%), indicated that strain CS1(T) represents a novel species of the genus Lactobacillus for which the name Lactobacillus rossii sp. nov. is proposed. The type strain of this species is CS1(T) (=ATCC BAA-822(T)=DSM 15814 T).