A significant decrease in pH occurred in the culture medium when cells of Selenomonas ruminantium HD-4 were incubated in the presence of relatively high concentrations of glucose (0.4 and 1.0%). Forty microM lasalocid reduced cell growth to 35.5 and 35.7% of control growth, respectively, for 0.05 and 0.4% glucose, while growth was completely inhibited by 40 microM lasalocid in the presence of 1.0% glucose. In the presence of 80 microM lasalocid, cells were unable to grow within 24 hr at any glucose concentration. In the case of treatment with 10 microM lasalocid, growth was inhibited when pH decreased below 6.0 in the presence of 0.4 and 1.0% glucose. With 0.05% glucose, pH never reduced to below 6.4 within 24 hr and there was no significant effect of lasalocid on either growth or pH. When cultures were treated with 10 microM lasalocid at the mid-exponential phase, growth was moderately inhibited and abnormal cells which had outer membrane detached from the inner membrane layer resulting in cell lysis were found in thin sections. These results indicate that the presence of large amounts of glucose decreases pH in culture medium and that the decrease in pH to below about 6.0 causes the increased effect of lasalocid on the growth of S. ruminantium. These results suggest that the antimicrobial activity of lasalocid is potentiated by a decrease in extracellular pH.