Measuring the concentration of the pharmacologically active metabolite of mycophenolic acid (MPA), acyl-MPAG (AcMPAG), in addition to the pharmacologically inactive phenol glucuronide metabolite (MPAG) may prove useful in the therapeutic drug monitoring of MPA. A simple high-performance liquid chromatography method with ultraviolet detection (HPLC-UV) was established for simultaneous determination of MPA, AcMPAG, and MPAG in human plasma. The method utilizes 2 internal standards (IS), phenolphthalein glucuronic acid (PGA) for MPAG and a carboxy butoxy derivative of MPA (MPAC) for AcMPAG and MPA. The method consists of solid-phase extraction of the analytes followed by analysis over a Zorbax Rx C8 column (150 x 4.6 mm, 5 mum) at 254 nm. The analytes were separated with a gradient mixture of methanol and 0.1% phosphoric acid over a run time of 14 minutes at a flow rate of 1 mL/min. The assay was linear in the concentration range from 0.2 to 50 mg/L for MPA, 0.5 to 25 mg/L for AcMPAG, and 2 to 500 mg/L for MPAG. The mean +/- SD interday accuracy and %CV for MPA were 100.3 +/- 5.7 and 5.7%, for AcMPAG, 102.6 +/- 5.7 and 5.6%, and for MPAG 100.5 +/- 5.3 and 5.3%, respectively. The average +/- SD of MPA, MPAG, and AcMPAG maximum concentrations (Cmax) in 23 kidney transplant recipients on 500 or 1000 mg twice daily mycophenolate mofetil were 11.77 +/- 9.43, 88.15 +/- 46.4, and 3.01 +/- 1.73 mg/L, respectively, and the predose trough (Cmin morning) concentrations were 2.24 +/- 3.11, 55.44 +/- 29.55, and 1.42 +/- 0.74 mg/L, respectively. The method described is robust, sensitive, reproducible, and will be useful in therapeutic drug monitoring or pharmacokinetic studies of MPA.