The phylogenetic positions of 40 Butyrivibrio strains were determined by performing a comparative sequence analysis of the 16S rRNA genes of these organisms. We found that all of the strains which we studied belong to cluster XIVa (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez=Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) of the Clostridium subphylum of the gram-positive bacteria, which also includes several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species. We also found that the Butyrivibrio strains which we examined were genotypically heterogeneous and exhibited 12 distinct rRNA sequence types. The 12 rRNA sequence types formed three distinct lineages in cluster XIVa, which were separate from each other and from all other species belonging to this cluster. One lineage consisted of strains which exhibited a single rRNA type and corresponded to the species Butyrivibrio crossotus. The second lineage consisted of 12 strains designated Butyrivibrio fibrisolvens which exhibited seven distinct rRNA sequence types. The type strain of B. fibrisolvens was a member of this lineage, but its position was peripheral. The third lineage comprised 26 B. fibrisolvens strains which exhibited four distinct rRNA sequence types. Tree topology and sequence divergence considerations indicated that the three lineages correspond to three separate genera and that the genus Butyrivibrio should be restricted to the group that contains the type strain of B. fibrisolvens.