The in vitro metabolism of strychnine was studied in the 9000g supernatant fractions from rat and rabbit livers. The metabolism was markedly inhibited by cytochrome P-450 inhibitors, SKF-525A and n-octylamine, but only slightly by a microsomal FAD-containing monooxygenase inhibitor, methimazole. Five metabolites formed in vitro with rabbit liver were isolated and purified by Sep-Pak C18 cartridge chromatography and preparative TLC. Three of them were identified as 2-hydroxystrychnine, strychnine N-oxide, and 21 alpha, 22 alpha-dihydroxy-22-hydrostrychnine by comparison with their authentic samples by means of UV, NMR, and mass spectrometries. An additional two metabolites were tentatively identified as strychnine 21,22-epoxide and 11,12-dehydrostrychnine by spectral measurements. Four of these metabolites, with the exception of 2-hydroxystrychnine, were novel metabolites of strychnine. The in vitro formation of these metabolites by rabbit liver was determined by HPLC after partial purification. The major identified metabolite was strychnine N-oxide, which accounted for approximately 15% of the metabolized strychnine. All the other metabolites accounted for less than 1%. The presence of a larger quantity of other metabolites which have been neither isolated nor identified was also suggested.