Two bacterial strains, DSM 20253T and DSM 20254, isolated from rennin and regarded as causing cheese spoilage, were deposited in the DSMZ as Lactobacillus sp. by J. Stadhouders. The strains show 99.9% 16S rRNA gene sequence similarity and have less than 94.3% similarity with any other species of the genus. Lactobacillus coryniformis is their closest phylogenetic neighbour. DNA-DNA hybridization experiments confirmed that the two strains are members of the same species with separate status within the genus Lactobacillus. The strains are homofermentative lactic acid bacteria and can be phenotypically and genotypically distinguished from their closest relatives. 16S rRNA gene-targeted specific primers were designed to enable PCR detection of the novel species. The name Lactobacillus rennini sp. nov. is proposed for the novel isolates, with strain CECT 5922T (=DSM 20253T) as the type strain.