The metabolic formation of α-hydroxytamoxifen, a reactive metabolite of tamoxifen in rat liver, was characterized and quantified in vitro (hepatic microsomal incubations) and in vivo (bile-duct cannulated animals). This minor metabolite was identified by chromatographic and mass spectral comparisons with the authentic compound. The rates of formation of α-hydroxytamoxifen in incubations (30 min) of tamoxifen (25 μM) with liver microsomal preparations from women (pool of six), female CD1 mice or female Sprague–Dawley rats, as quantified by liquid chromatography–mass spectrometry (LC–MS), were 1.15 ± 0.03, 0.30 ± 0.05 and 2.70 ± 0.35 pmol/min/mg protein, respectively. Selective inhibition of microsomal P450 indicated that α-hydroxylation was catalysed predominantly by CYP3A in humans. Bile-duct cannulated and anaesthetized female rats and mice given [¹⁴C]tamoxifen (43 μmol/kg, i.v.) excreted, respectively, 24 and 21% of the administered radioactivity in bile over 5 and 3.5 h. The major radiolabelled biliary metabolite in rats, characterized by LC–MS after enzymic hydrolysis of conjugates, was the glucuronide of 4-hydroxytamoxifen (10% of dose) and only 0.1% of the dose was recovered as α-hydroxytamoxifen. After administration of α-hydroxytamoxifen (43 μmol/kg, i.v.) to rats, only 1.19% of the administered compound was recovered from a glucuronide metabolite in bile, indicating a possible 0.84% α-hydroxylation of tamoxifen in vivo. There was, however, no indication of the presence in bile of either O-sulphonate or glutathione conjugates derived from α-hydroxytamoxifen. This study shows for the first time that α-hydroxytamoxifen can be glucuronylated in rat liver. Whereas sulphonation results in electrophilic genotoxic intermediates, glucuronidation may represent a means of detoxifying α-hydroxytamoxifen.