The non-cellulolytic ruminal bacterium, Prevotella bryantii, grew rapidly on water soluble β-glucans, but a mutant deficient in carboxymethylcellulase (CMCase) activity could not. Native activity gels prepared with CMC and β-glucan indicated that the CMCase and β-glucanase activities migrated at similar rates. When a plasmid carrying the P. bryantii CMCase was transferred to Escherichia coli, the clone had CMCase and β-glucanase activities. P. bryantii grew on mixed β-1,3-1,4 glucans, but not on β-1,3 glucan, and similar results were obtained with the cellulolytic ruminal bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus B199). Mixed ruminal bacteria from cattle fed hay had twice as much CMCase activity as bacteria from cattle fed 90% cereal grain (P<0.05), and the CMCase and β-glucanase activities were highly correlated (r2 = 0.93) for the 22 samples tested. The CMCase and β-glucanase activities of mixed ruminal bacteria migrated slowly through polyacrylamide gels, but the migration distances were approximately the same. When β-glucan-utilizing ruminal bacteria were isolated from cattle fed hay or 90% cereal grain, 70 and 38% of the strains, respectively, had CMCase activity. A similar trend was observed with cellobiose-utilizing isolates (70 and 35%, respectively, were CMCase positive). All CMCase positive, cellobiose-utilizing ruminal bacteria could grow on β-glucan. CMCase activity was not strongly correlated with cellulose utilization, and less than 15% of the CMCase positive isolates grew on ball-milled cellulose. Based on these results, the cell-associated CMCases of ruminal bacteria provide a mechanism for utilizing water soluble β-glucans from cereal grains.