The in vitro metabolism of rosiglitazone (RGZ) was studied in freshly isolated human, rat, and monkey hepatocytes. The metabolites of [¹⁴C]RGZ produced by incubation with hepatocytes were detected by radioactivity detection high-performance liquid chromatography. Seven metabolites (M1-M7) of RGZ were detected in human hepatocytes. The structures of the metabolites were elucidated by liquid chromatography/tandem mass spectrometry using electrospray ionization. The structural analysis demonstrated that M1, M2, and M4 were novel metabolites of RGZ. M1 was identified as a 2,4-thiazolidinedione (TZD) ring-opened N-glucuronide. M2 was identified as a TZD ring N-glucuronide. M4 was proposed to be a TZD ring-opened methylmercapto amide. Similarly, these metabolites were also detected in rat and monkey hepatocytes. To our knowledge, this is the first report on N-glucuronidation of TZD rings. Based on the structures of the metabolites, we propose the following novel in vitro metabolic pathways for RGZ: 1) N-glucuronidation of the TZD ring of RGZ to form M2 followed by hydrolysis to the TZD ring-opened N-glucuronide M1; and 2) methylation of the mercapto group of the TZD ring-opened mercapto amide to form M4.