A high-performance liquid chromatography (HPLC) method has been developed for the determination of morphine and its main metabolites, morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G), in plasma or cerebrospinal fluid. Samples were extracted using on-line solid-phase extraction followed by reversed-phase HPLC with fluorescence detection. Recoveries of 20 ng morphine and morphine glucuronides in plasma were over 95%. The limit of detection using 400 microliters of a biological matrix was 0.85, 3.4 and 1.0 ng/ml of M-3-G, M-6-G and morphine, respectively. Inter- and intra-day assay precision was better than 10%. The main advantages of the present described method are increased recoveries (> 95%) and a high degree of automation allowing a high speed in routine analysis. The time required for the fully automated analysis of one sample was less than 26 min.