Previous mass balance studies in humans and mice have shown that the fecal and urinary recovery of paclitaxel and known metabolites (3' -hydroxypaclitaxel, 6alpha-hydroxypaclitaxel and 3',6alpha-dihydroxypaclitaxel) was not complete. Obviously this discrepancy is caused by the existence of other yet unknown metabolites. Mdr1a/1b(-/-) mice excrete very low quantities of unchanged paclitaxel. We have therefore used these mice receiving i.v. [3H]paclitaxel to further study the metabolic fate of paclitaxel. The major part of the radiolabel, being 70%, was excreted in the feces. A lipophilic sample, containing about 70% of the radioactivity present in the feces sample, was obtained by diethyl ether extraction. The aqueous residue containing about 30% of the radioactivity was further extracted using methanol. The high-performance liquid chromatography (HPLC) chromatograms of the lipophilic and aqueous sample revealed two and five putative new metabolites of paclitaxel, respectively. The HPLC fractions containing substantial amounts of radioactivity were subjected to tandem mass spectrometry. Two novel monohydroxylated paclitaxel structures were identified, which are probably 2m-hydroxypaclitaxel and 19-hydroxypaclitaxel, structures previously identified in rats. Including these metabolites, about 60% of the mass balance of paclitaxel could be quantified.