The objectives of this study were to examine the effects of chlorhexidine diacetate on growth and L-lactate production by Streptococcus bovis JB1 as well as the effects of this antimicrobial compound on the mixed ruminal microorganism fermentation. Addition of 1.8 mu M chlorhexidine diacetate to glucose medium resulted in a lag in growth by S. bovis JB1, and growth was completely inhibited in the presence of 3.6, 9.0, and 18 mu M chlorhexidine. When 6.2 mu M chlorhexidine diacetate was added to glucose medium after 2 h of incubation, glucose utilization and L-lactate production by S. bovis JB1 were reduced. Phosphoenolpyruvate-dependent phosphorylation of C-14-glucose by toluene-treated cells of S. bovis JB1 was inhibited by increasing concentrations (1.8 to 18 mu M) of chlorhexidine, whereas only the 18 mu M concentration reduced the membrane potential (Delta Psi). Chlorhexidine diacetate was a potent inhibitor of L-lactate and methane production from glucose fermentation by mixed ruminal microorganisms, However, because chlorhexidine also decreased acetate and propionate concentrations and increased ammonia concentrations in mixed-culture incubations, this antimicrobial compound may have limited application as a ruminant feed additive.