BACKGROUND AND OBJECTIVES Fentanyl has been used for cancer pain in transdermal formulation. The aim of the present study was to establish an analytical method for fentanyl in human plasma and in an applied transdermal reservoir patch (Reservoir-TTS), as well as for therapeutic monitoring of fentanyl in cancer patients. METHOD Electro-spray ionization mass spectrometric (ESI-MS/MS) analysis followed solid phase extraction (SPE) from human plasma and drug reservoir extraction from an applied Reservoir-TTS. Each separation was completed within 9 min using an ODS column (particle size, 3 microm, 2.0 mm i.d. x 75 mm) with 25% acetonitrile containing 5 mm ammonium acetate at pH 3.5. In the ESI-MS/MS analysis, the calibration curve for fentanyl was linear over a concentration range of 0.05-7.2 ng/mL in human plasma. The extraction efficiency of fentanyl in the human plasma was more than 95%. The intra- and interassay precision and accuracy were within 7% and 97.3-101.2%, respectively. The lower LOQ for fentanyl was 0.05 ng/mL in the human plasma. The extraction of the 25 microg/h and 50 microg/h Reservoir-TTS gave reproducible recoveries of 88.3% and 90.9%, respectively. The plasma concentration of fentanyl showed large interindividual variation in 31 patients with cancer pain. CONCLUSION The method described is simple, accurate, and reproducible, and should be helpful for the therapeutic monitoring of fentanyl in cancer patients.