A sensitive method for the determination of leuprorelin (TAP-144), a luteinizing hormone-releasing hormone analogue, and its C-terminal metabolite, M-I, in serum and urine has been developed. Leuprorelin and M-I were extracted from serum or urine samples with Sep-Pak C18 cartridges, and separated completely by high-performance liquid chromatography and determined by radioimmunoassay using [125I]leuprorelin as the labelled antigen. The detection limit of the method was 0.05 ng/ml for leuprorelin and M-I, and the recovery of the compounds added to serum and urine was over 88% with a coefficient of variation (within-assay) of less than 5%. The method was applied to the determination of leuprorelin and M-I-like immunoreactivity in serum or urine after administration of once-a-month injectable microspheres of leuprorelin acetate (TAP-144-SR) to patients with prostate cancer.