The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, intermediate in rats, chicks and pigs, and lowest in horses. When expressed as turnover number (nmol of metabolized monensin/min nmol cytochrome P450-1), the catalytic efficiency (chick >> cattle >> pig approximately rat > horse) was found to correlate inversely with the well known interspecies differences in the susceptibility to the toxic effects of the ionophore, which is characterized by an oral LD50 of 2-3 mg/kg bodyweight (bw) in horses, 50-80 mg/kg bw in cattle and 200 mg/kg bw in chicks. Chick and cattle microsomes also displayed both the highest catalytic efficiency toward two P450 3A dependent substrates (erythromycin and triacetyloleandomycin) and the highest immunodetectable levels of proteins cross-reacting with anti rat P450 3A1/2. Further studies are required to define the role played by this isoenzyme in the oxidative biotransformation of the drug in food producing species.