Aminopyrine administered to normal human volunteers in an oral dose of either 9 mg/kg or 4.5 mg/kg prolonged the plasma half-life and reduced the metabolic clearance rate of antipyrine (18 mg/kg, orally) without changing its apparent volume of distribution. By contrast, this same oral dose of antipyrine given simultaneously with 9 mg/kg aminopyrine failed to alter aminopyrine disposition. Thus, antipyrine and aminopyrine should not be administered simultaneously to measure different steps in hepatic drug oxidation, although in man aminopyrine can be given for this purpose 24 hr after antipyrine. Antipyrine elimination was prolonged to the same extent when aminopyrine was given 5 hr before antipyrine as when the drugs were given simultaneoulsy. Since in man aminopyrine has a biologic halic-life of approximately 2.7 hr, the marked inhibitory effects observed 5 hr after aminopyrine administration may be due to its major metabolite, 4-aminoantipyrine. To define mechanism by which aminopyrine affects antipyrine disposition in vivo, hepatic microsomes were prepared from rats, mice, and dogs, and rates of antipyrine hydroxylation were measured in vitro both in the absence and in the presence of aminopyrine. In these species in vitro inhibition of antipyrine hydroxylation by 4-aminoantipyrine was of a mixed type; antipyrine inhibited competitively aminopyrine N-demethylation in vitro in rats, mice, and dogs. There were some sex and species differences in the Km' V max' and Ki for aminopyrine and antipyrine.