In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.The species Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum are genotypically closely related and show highly similar phenotypes. The genetic heterogeneity of the L. plantarum group has been demonstrated by Dellaglio et al. (8) on the basis of DNA-DNA hybridization data: three groups were identified which were later classified as L. plantarum sensu stricto (2), L. pentosus (28), and L. paraplantarum (7).Despite the importance of these species for fermented foods of plant, animal, and fish origin, their correct identification is complicated by the ambiguous response of traditional physiological tests and molecular methods: Fourier transform infrared spectroscopy of lactobacilli from breweries was not able to differentiate spectra from L. plantarum and L. pentosus (6). Randomly amplified polymorphic DNA-PCR and related numerical analysis gave satisfying results, but the methods were applied only to L. plantarum and L. pentosus (27). Finally, two papers reported the selection of species-specific PCR primers for the L. plantarum group; however, in one case specificity towards L. paraplantarum was not checked (21), and in the other the primers did not guarantee sufficient specificity (3). Satisfying results have been obtained by Southern-type hybridization with a pyrDFE probe (4), randomly amplified polymorphic DNA-PCR, and AFLP (Keygene NV, Wageningen, The Netherlands) (S. Torriani, F. Clementi, F. Dellaglio, B. Hoste, M. Vancanneyt, and J. J. Swings, Proc. Sixth Symp. Lactic Acid Bacteria, poster B2, 1999), but such methods are not suitable for routine identification requirements. The difficulty of correct identification of these species and the increasing interest in some of their properties, e.g., probiotic activities (13) and tannin degradation (19), indicates the need for a simple and reliable molecular method for the definite differentiation of L. plantarum, L. parap...