The aim of this study was to characterize the metabolic pathways of 2-methoxyestradiol (2ME2), an investigational anticancer drug. In vitro metabolism studies were performed by incubation of 2ME2 with human liver microsomes under various conditions and metabolite identification was performed using liquid chromatography-tandem mass spectrometry. In microsomal mixtures, four major oxidative metabolites and two glucuronic acid conjugates were observed originating from 2ME2. Human liver S9 protein fraction was used to screen for in vitro sulfation but no prominent conjugates were observed. The total hepatic clearance as estimated using the well-stirred model was approximately 712 mL/min. In vivo metabolism, assessed using 24-h collections of urine from cancer patients treated with 2ME2 revealed that