Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.