Selected phenotypic characteristics of isolates of Prevotella ruminicola (formerly Bacteroides ruminicola) were studied in order to establish whether the characteristics of genotypic strain groups established previously on the basis of 16S ribosomal DNA sequences differed systematically. Among strains formerly considered P. ruminicola subsp. brevis, strains related to strain GA33T (T = type strain) typically failed to produce carboxymethyl cellulase (CMCase) activity detectable by plate assays and failed to ferment xylose, while strains related to strain B14T produced abundant CMCase and fermented xylose. We propose that strains related to GA33T, which have DNA G + C contents between 45 and 51 mol%, should be assigned to a new species, Prevotella brevis, and that strains related to B14T, which have DNA G + C contents between 39 and 43 mol%, should be assigned to another new species, Prevotella bryantii. Most of the isolates formerly classified as P. ruminicola subsp. ruminicola strains produced CMCase and had DNA G + C contents between 45 and 51 mol%, and we propose that these organisms should be placed in the redefined species P. ruminicola. A small group of isolates that have lower G + C contents are assigned to another new species, Prevotella albensis. Most P. brevis and P. bryantii strains produced abundatn extracellular DNase activity. Proteinase activities (as determined by [14C]casein hydrolysis) varied widely between strains, and P. brevis strains exhibited the highest mean activity. All strains produced dipeptidyl peptidase activity, but the relative activities against different peptide substrates exhibited by P. bryantii, P. albensis, and P. brevis differed systematically. The phenotypic differences among the newly defined species suggest that they may occupy distinct niches within the rumen ecosystem.