A quantitative analysis of prostaglandin-related substances has been developed. Hydroxyl groups of prostaglandin and thromboxane were acetylated by acetic anhydride, the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, twenty kinds of prostaglandin derivative could be detected simultaneously within 11 min on a selected-ion monitoring detection chromatogram without a gradient system. Generally, the base ion, [M + H - n(60)]+, is produced through elimination of acetic acid (n = number of the hydroxyl group of prostaglandin or thromboxane). The detection limit for these derivatives was ca. 0.2 pmol at the levels of prostaglandin-related substances prior to derivatization. They could be analysed in the range 0.5-10 pmol. The assay was successfully applied to prostaglandin-related substances in human seminal fluid and rat brain.