The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys. In metabolization using liver microsomes, the TZD ring-opened mercapto amide (M22) and TZD ring-opened mercapto carboxylic acid (M23) were identified as the primary metabolite of the TZD ring-opening pathway and its sequential metabolite, which have not been detected previously from in vivo studies. Combination with S-adenosyl-L-methionine was useful to obtain the sequential S-methylated metabolites from the oxidative metabolites. N-Glucuronide and sequential TZD ring-opened metabolites were also found in liver microsomes in the presence of UDP-glucuronic acid. The O-demethyl-O-sulfate (M11), which is the major in vivo metabolite in rats and monkeys, was detected in all species of hepatocytes. In addition, a TZD ring-opened S-cysteine conjugate (M15) was detected in human hepatocytes. From these results, the in vivo metabolic pathways in humans were predicted to be the four oxidation and one N-glucuronidation pathways. The four oxidative metabolites were formed by multiple human P450 enzymes, and N-glucuronide was formed by UGT1A3 and UGT2B7.