In humans, approximately two-thirds of the metabolic clearance of ziprasidone proceeds through reduction of the N-S bond of the benzisothiazole moiety, which is followed by methylation of the resulting thiophenol to the major metabolite S-methyldihydroziprasidone. The objective of this study was to gain an understanding of the underlying mechanism of this clearance route. Incubation of ziprasidone in human liver cytosol yielded the reduction product dihydroziprasidone. Heating the cytosol prior to incubation prevented the reaction, and the reaction was saturable (K(M) = 3.9 µM; V(max) = 0.24 nmol/min/mg protein), supporting that it was enzyme catalyzed. It was partially stimulated by 2-hydroxypyrimidine and inhibited by menadione, supporting a role for aldehyde oxidase in this reaction. However, incubation of ziprasidone with reduced glutathione, even in the absence of cytosol, readily yielded dihydroziprasidone indicating that there is also a chemical reduction component to this clearance pathway. The pathway was reversible because incubation of dihydroziprasidone in cytosol or with oxidized glutathione in buffer yielded ziprasidone. The methylation of dihydroziprasidone was observed in human liver microsomes in the presence of S-adenosylmethionine (K(M) = 14 µM; V(max) = 0.032 nmol/min/mg protein). This reaction was inhibited by 2,3-dichloro-α-methylbenzylamine supporting that thiol methyltransferase is the enzyme responsible for this reaction. Thus, the main metabolic pathway for ziprasidone in humans occurs via chemical reduction and aldehyde oxidase catalyzed reduction, followed by thiol methyltransferase catalyzed methylation.