Streptococcus bovis, a Gram-positive ruminal bacterium, was unable to grow in the presence of monensin. When monensin (5 mg/liter) was added to actively growing cultures, there was an immediate decrease in growth rate, and within 3 h no further growth was observed. Glucose utilization and lactate production continued for another 8 h even though growth had ceased. Monensin caused a decrease (P less than .05) in intracellular K+, a decrease (P less than .05) in intracellular pH and an increase (P less than .05) in intracellular Na+. The net exchange of K+ for Na+ and H+ via monensin was driven by the difference in concentration of K+ and Na+ across the cell membrane. Non-treated cells maintained a 70-fold gradient (inside higher) for K+, while the Na+ gradient was only 2.7-fold (inside higher). Previous models were based on a reverse mechanism whereby monensin would drive an efflux of Na+ from ruminal bacteria, and were not supported by experimental measurements of intracellular Na+ and K+.