The objective of this study was to compare the effects of laidlomycin propionate and monensin on cell growth, glucose fermentation, and glucose uptake in Streptococcus bovis strain JB1 and Selenomonas ruminantium strain HD4. Experiments were also conducted to compare the effects of both ionophores on sodium-dependent serine transport and cell yield in S. bovis. Batch cultures (500 mL) of each bacterium were grown on 3.6 g/L D-glucose in semidefined medium and treated with either 5 ppm monensin or 2 ppm laidlomycin propionate (n=2). Cell growth was monitored by measuring optical density at 600 nm (OD600). Glucose and L-lactate concentrations were measured using coupled enzyme assays. In S. bovis, both monensin and laidlomycin propionate decreased OD600, glucose utilization, and L-lactate production. Neither ionophore had any effect on glucose utilization by S. ruminantium. [14C]Glucose uptake between 5 and 30 min by both bacteria was not altered by either ionophore. Sodium-dependent [14C]serine uptake by S. bovis was inhibited by monensin but not laidlomycin propionate. When S. bovis was grown in glucose-limited continuous culture (dilution rate=.10 h(-1)) at extracellular pH 6.7, increasing concentrations of both ionophores decreased bacterial yield, and both ionophores were more potent at an extracellular pH of 5.7. However, monensin was a more potent inhibitor than laidlomycin propionate at pH 6.7 and 5.7. Collectively, these results suggest that the ionophore laidlomycin propionate inhibits the Gram-positive bacterium S. bovis in a manner similar to that of monensin, but, at the concentrations used in this study, laidlomycin propionate seems to be less potent than monensin in inhibiting serine uptake and cell yield.