The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase was investigated. The existence of a free radical intermediate formed during enzymatic oxidation was supported by a ratio of hydrogen peroxide: p-phenetidine consumed of 1:2 in the horseradish peroxidase system. Furthermore in both enzyme systems a rapid oxidation of added glutathione was observed and in the presence of the thiol there was a decreased removal of p-phenetidine. This suggests the reduction of a p-phenetidine radical by glutathione generating p-phenetidine and a thiyl radical. The latter react with oxygen and a rapid oxygen uptake was observed during enzymic oxidation in the presence of thiols. That p-phenetidine radicals were produced during horseradish peroxidase catalyzed oxidation of p-phenetidine was supported by experiments using the spin probe OXANOH. This was oxidized to its stable free radical form (OXANO.) in an enzyme- and substrate-dependent reaction and the EPR signal obtained was not decreased by SOD (80 micrograms/ml) or benzoate (10-100 mM). TLC characteristics of the products of the oxidation of p-phenetidine by both enzymes were almost identical inferring a similar mechanism of oxidation. Two of the metabolites were characterized by mass spectrometry and by comparison with reference compounds prepared by chemical oxidation. One metabolite was identified as 4,4'-diethoxyazobenzene, which further supports a radical mechanism, and the other was a p-phenetidine trimer which could exist in both oxidized and reduced forms. On the basis of these observations a mechanism for the oxidation of p-phenetidine and the fate of glutathione during such oxidations is proposed.