The polyether ionophore, laidlomycin, and several acyl derivatives were tested for their ability to favorably alter fermentation in two types of in vitro rumen fluid incubations. Dose response data were used to estimate the concentration (microgram/ml) of each ionophore required for either 50% maximal enhancement of propionic acid production (EC50) or 50% maximal inhibition of lactic acid production (IC50). Acylation of laidlomycin with straight-chain acyl groups from two to 12 carbon atoms tended to improve the potency of laidlomycin, especially for inhibiting lactic acid production. Comparative incubations using laidlomycin butyrate, laidlomycin and monensin indicated that both laidlomycin butyrate (EC50 = .3) and monensin (EC50 = .7) were more potent enhancers of propionic acid production than laidlomycin (EC50 = 2.0; P less than .05). Laidlomycin butyrate (IC50 = .3) was a more potent inhibitor of lactic acid production than either laidlomycin (IC50 = 1.8) or monensin (IC50 = 1.3; P less than .05). In a continuous culture experiment, three chemostats each received laidlomycin butyrate or monensin at the rate of .5 micrograms/ml of effluent/d while two flasks remained as control. Propionic acid production was increased (P less than .01) from 22.9 mmol/d in control flasks to 30.5 and 33.7 mmol/d in flasks treated with monensin and laidlomycin butyrate, respectively. Concomitant decreases in production rates of acetic, butyric and isovaleric acids also were observed (P less than .01). Thirty-six steers were used in a 56-d trial to evaluate effects of laidlomycin butyrate and monensin, at 33 mg/kg of diet, on feedlot performance.(ABSTRACT TRUNCATED AT 250 WORDS)