The N-reduction of the centrally acting alpha 2-adrenoreceptor agonist guanoxabenz (Benzérial), an N-hydroxyamidinohydrazone, to the amidinohydrazone guanabenz (Wytensin, Hipten, Rexitene) by microsomal fractions from rabbits, pigs and humans has been detected in vitro. The conversion rates with rabbit microsomal fractions were markedly slower than those in the cases of fractions from humans and pigs. It was also possible to demonstrate the N-oxidation of guanabenz to guanoxabenz by the use of microsomal fractions from rabbits, pigs, and humans. Furthermore, the oxidation was also observed in reconstituted systems in the presence of enriched cytochrome P450 fractions, purified isoenzyme P450 2C3, and heterologously expressed P450 2C3 of the subforms 6 beta H and 6 beta L. The analyses were performed with a newly developed HPLC technique and were confirmed by LC-MS methods. The kinetic parameters determined for the metabolic cycle (bioreversible reactions) are indicative of a predominance of the reduction of guanoxabenz to guanabenz in vivo. Accordingly, guanoxabenz in part constitutes a prodrug of guanabenz. Examination of guanabenz and guanoxabenz for mutagenicity by means of the Ames test revealed that guanoxabenz has pronounced mutagenic effects in the strains TA 98 and TA 1537. Guanabenz did not exhibit mutagenicity so that the N-reduction of guanoxabenz has significance in terms of detoxification.