Epothilones are natural macrolides displaying potent antiproliferative properties against various cell lines and capable to bind tubulin and acting as microtubule-stabilizing agents like taxoids. We intended to isolate and characterize epothilone metabolites and identify enzymes implicated in the biotransformation process. In the presence of NADPH, liver microsomes from phenobarbital-treated rats produce two metabolites resulting from the oxidation of either epothilone A or B by CYP isoforms. Similarly, the oxidative biotransformation of epothilones A and B by human liver microsomes generates three metabolites with Km values ranged from 61 to 86µM. The two major metabolites (m1 and m2) are hydroxylated on the macrolide ring essentially by CYP3A4, whereas 3A5, 3A7 and 2B6 are minor contributors to the reaction. M3 is formed by CYP2C19 and 2C9 and results from the hydroxylation of the methyl on carbon 17 of the lateral chain. Inhibition of CYP3A almost completely abolished the formation of m1 and m2, whereas inhibition of CYP2C19 substantially reduced the production of m3. Collectively these data suggest that the oxidative metabolism of epothilones is principally mediated by CYP3A4 and CYP2C19. Epothilone B was found to be an in vitro inhibitor of CYP2C9 [IC₅₀≈25µM], CYP2C19 [Ki≈1.7µM] and CYP3A4/5 [K_i≈1.85µM] whereas conversely taxanes or Vinca alkaloids significantly reduced oxidation of epothilone B. However, clinically relevant inhibition in patients undergoing chemotherapy is unlikely due to low therapeutic epothilone B blood concentrations.