To characterize a phase 1 metabolite of mycophenolic acid (MPA) and the human cytochrome P450 isoform(s) (CYP) involved in its formation. MPA metabolites were investigated in blood and urine samples from transplant patients under mycophenolate mofetil therapy (n = 5) as well as with in vitro incubation of MPA with human liver microsomes. The CYP isoforms involved in the oxidative metabolism were investigated in vitro on human liver microsomes with isoform-specific inhibitors as well as in human embryonic kidney cell lines expressing recombinant human CYPs. The analytic methods used were based on LC-MS/MS. A 6-O-desmethyl-MPA (DM-MPA) metabolite and 2 related glucuronides were identified in patients' blood and urine. Human liver microsomes produced DM-MPA with an apparent Km = 0.83 +/- 0.06 mmol/L and Vmax = 5.57 +/- 0.29 pmol/mg/min. The CYP3A inhibitor ketoconazole was found to inhibit DM-MPA formation by 50.3% with respect to the control, and trimethoprim (CYP2C8 inhibitor) reduced it by 30.1%. However, DM-MPA was produced only by the transfected cell lines expressing CYP3A4 and, to a lesser extent, CYP3A5. In vitro, MPA at concentrations above the plasma therapeutic range was found to decrease the metabolism of tacrolimus, suggesting a possible competition for CYP3A. No effect of MPAat therapeutic or higher level was found on cyclosporin metabolism. The phase 1 metabolite of MPA previously known as M-3 was identified as 6-O-desmethyl-MPA and is produced by CYP3A4/5 and probably CYP2C8. MPA might compete with other drugs on CYP3A because of its high therapeutic concentrations, although this was not the case for cyclosporin and to only a small extent for tacrolimus.