Species differences in oral bioavailability, first-pass metabolism and pharmacokinetics of biopharmaceutics classification system (BCS) class I compound acetaminophen were studied. The absolute bioavailability was 42.2%, 39.0%, 44.5%, 75.5% and 91.0% in chickens, turkeys, dogs, pigs and horses, respectively. After hydrolysis of metabolites by beta-glucuronidase/sulfatase, apparent bioavailability increased significantly in all species (turkeys: 72.4%, dogs: 100.5%, pigs: 102.2%), except horses (91.6%). Mean metabolic ratios of [acetaminophen glucuronide]/[acetaminophen] between 0 and 1h were significantly higher after oral dosing in turkeys, dogs and pigs, revealing the role of first-pass metabolism in incomplete bioavailability. Evidence of species differences in acetaminophen metabolism is provided by differences in plasma clearance, which was inversely proportional to bioavailability. In conclusion, differences in BA appeared to originate predominantly from differences in first-pass metabolism, demonstrating that the BCS high permeability classification of acetaminophen is consistent across the mammalian species studied. In turkeys, however, incomplete absorption additionally seemed to contribute to the low BA.