A highly specific method, selected ion monitoring (SIM), using [7,7-2H2] 19-hydroxytestosterone (19-OHT) as an internal standard, was newly developed to determine 19-OHT in serum. Steroids in serum obtained from the ovarian vein were extracted with ethyl acetate. Following separation by thin layer chromatography, the 19-OHT fraction was subjected to SIM after formation of the methoxime-trimethylsilyl (MOTMS) derivative. Mass spectra of the 19-OHT-MOTMS derivative was dominated by the molecular cation [M] + (m/e 477). Serum concentration of 19-OHT was 280 pg/ml in ovarian vein. This is the first evidence of 19-OHT in human blood.