Isaxonine phosphate or Nerfactor (2-isopropylaminopyrimidine) has been implicated in several cases of hepatitis which is reversible after withdrawal of the drug. In order to understand the cause of such hepatitis, the metabolic activation of isaxonine phosphate with different liver microsomes was investigated. The major metabolites were 5-hydroxyisopropylaminopyrimidine and 2-aminopyrimidine. Covalent binding to microsomal proteins was also detected. In vitro metabolic activation required intact microsomes, NADPH and O2 as cofactors and was cytochrome P-450 dependent. A sensitive fluorimetric assay of 5-hydroxyisaxonine was developed. The metabolism of isaxonine phosphate was compared in liver microsomes from rat, rabbit, dog, monkey and man and found to be qualitatively similar. Treatment of rats with phenobarbital increased the formation of 5-hydroxyisaxonine, while treatment with 3-methylcholanthrene increased the formation of 2-aminopyrimidine but decreased that of 5-hydroxyisaxonine. Inhibition and reconstitution experiments demonstrated that 5-hydroxylation of isaxonine was catalyzed by a cytochrome P-450. Metabolic oxidation of isaxonine phosphate using 5-[3H]isaxonine phosphate led to a total loss of tritium in 5-hydroxyisaxonine and partial loss of tritium in 2-aminopyrimidine and covalent binding to proteins.