The characteristics of isoniazid amidase which hydrolyzes isoniazid to isonicotinic acid and hydrazine was examined in vitro using rat liver subcellular fractions. The activity of isoniazid amidase was estimated from the amount of hydrazine produced from a substrate, isoniazid, by means of GC-MS. High activity of the amidase was observed in the microsomal and lysosomal fractions, and at pH 7.4-7.8 in the microsomal fraction. The amidase was not inhibited by acetanilide, but by procaine and bis(p-nitrophenyl)phosphate. As expected, acetylisoniazid, a main metabolite of isoniazid, also inhibited the amidase. Not only the microsomal monooxygenase but also amidase was strongly induced by pretreatment with phenobarbital, 3-methylcholanthrene and rifampicin, respectively.