The metabolism of a new antisecretory and antiulcer drug, trithiozine (I.S.F. 2001, T), was studied in 4 hr rat urine samples after i.p. administration. After extraction at pH 7 with chloroform, the urine was either incubated with beta-glucuronidase or acidified to pH 2 and subsequently extracted with chloroform. The organic layers were evaporated to dryness and the residues used for TLC analysis. The neutral extracts revealed five spots, not present in control rat urine, corresponding to the unchanged drug T and to four metabolites. Two of the metabolites had been previously identified as the 4-(3,4,5-trimethoxybenzoyl)tetrahydro-1,4-oxazine (TBO) and the 4-(3,4,5-trimethoxythiobenzoyl)tetrahydro-1,4-oxazine-S-oxide (TO). Three other metabolites were found in the extracts after beta-glucuronidase incubation. TLC, U.V. and M.S. data were consistent with the structure 4-(3,5-dimethoxy-4-hydroxythiobenzoyl)tetrahydro-1,4-oxazine (HT), the corresponding S-oxide (HO) and the 4-(3,5-dimethoxy-4-hydroxybenzoyl)tetrahydro-1,4-oxazine (HBO). The acidic extracts revealed two spots structurally identified as the 3,4,5-trimethoxybenzoic acid (TBA) and the previous HBO. On the basis of present knowledge, a possible metabolic pathway of T is reported, consisting in a rapid metabolic oxidation on the sulfur atom and a slower demethylation on the para methoxy group. The presence of TBA is indicative of subsequent enzymatic hydrolysis of TBO. The intense and long-lasting inhibitory effect of T on gastric secretion is tentatively correlated with the pharmacological activities of some of its metabolites.