Incubation under air of [14C]tianeptine (0.5 mM) with a NADPH-generating system and hamster, mouse or rat liver microsomes resulted in the in vitro covalent binding of [14C]tianeptine metabolites to microsomal proteins. Covalent binding to hamster liver microsomes required NADPH and oxygen; it was decreased in the presence of the cytochrome P-450 inhibitors, carbon monoxide, piperonyl butoxide (4 mM), and SKF 525-A (4 mM) or in the presence of the nucleophile, glutathione (1 or 4 mM). In vitro covalent binding to hamster liver microsomes was not decreased in the presence of quinidine (1 microM), and was similar with microsomes from either female Dark Agouti, or female Sprague-Dawley rats. In contrast, in vitro covalent binding to hamster liver microsomes was decreased in the presence of troleandomycin (0.25 mM), while covalent binding was increased with microsomes from either hamsters, mice or rats pretreated with dexamethasone. Preincubation with IgG antibodies directed against rabbit liver glucocorticoid-inducible cytochrome P-450 3c(P-450 IIIA4) decreased in vitro covalent binding by 53 and 89%, respectively, with microsomes from control hamsters and dexamethasone-pretreated hamsters, and by 60 and 81%, respectively, with microsomes from control and dexamethasone-pretreated rats. We conclude that tianeptine is activated by hamster, mouse and rat liver cytochrome P-450 into a reactive metabolite. Metabolic activation is mediated in part by glucocorticoid-inducible isoenzymes but not by the isoenzyme metabolizing debrisoquine. In vivo studies are reported in the accompanying paper.