14C-Isoniazid (20 mg/kg po or iv) was administered alone or in combination with aspirin (100 mg/kg po), rifampin (30 mg/kg po), ethambutol (100 mg/kg po), or ethanol (3 g/kg po) to rats. In another experiment, phenobarbital sodium (40 mg/kg/day ip) was administered for 3 days prior to isoniazid. Aspirin and ethanol retarted the rate of isoniazid absorption from the GI tract. None of the drugs significantly altered the 14C-elimination rate from the blood over the first 4 hr. A tissue distribution study showed that changes in the blood levels produced by ethanol were reflected in the other tissues. When isoniazid was given intravenously, ethanol increased the amount of carbon-14 excreted in urine up to 24 hr after dosing; no other changes were observed in the total carbon-14 recovered in urine. Aspirin inhibited the conjugation of isonicotinic acid with glycine. Ethanol increased N-acetylisoniazid excretion and decreased isonicotinic acid excretion. None of the other treatments had more than a slight effect on isoniazid metabolism. Acute doses of isoniazid failed to produce any signs of hepatotoxicity, as judged by measurement of serum transaminase levels. The data do not suggest that any of the drugs studied are likely to potentiate the hepatotoxicity of isoniazid when administered acutely. Isoniazid metabolism in rats differed quantitatively from that reported for humans.