Urinary metabolites of boldenone (androsta-1,4-dien-17 beta-ol-3-one) following oral administration of boldenone (doses from 11 to 80 mg) to man were isolated from urine via XAD-2 adsorption and enzymatic hydrolysis with beta-glucuronidase from Escherichia coli. The isolated metabolites were derivatized with N-methyl-N-trimethylsilyltri- fluoroacetamide/trimethyliodosilane and analysed by gas chromatography/mass spectrometry with electron impact (EI) ionization at 70 eV. Boldenone (I) and four metabolites were identified after hydrolysis of the urine with beta-glucuronidase: 5 beta-androst-1-en-17 beta-ol-3-one (II), 5 beta-androst-1-ene-3 alpha, 17 beta-diol (III), 5 beta-androst-1-en-3 alpha-ol-17-one (IV) and 5 beta-androst-1-en-6 beta-ol-3,17-dione (V). Five further metabolites in low concentration were identified without enzymatic hydrolysis after treatment of the urine with potassium carbonate: 5 beta-androst-1-ene-3,17-dione (VI), 5 alpha-androst-1-ene-3,17-dione (VII), androsta-1,4-diene-3,17-dione (VIII), androsta-1,4-diene-6 beta,17 beta-diol-3-one (IX) and androsta-1,4-dien-6 beta-ol-3,17-dione (X). The identification of the metabolites is based on the gas chromatography retention index, high-performance liquid chromatography retention, EI mass spectrum, chemical reactions of the isolated metabolites, and synthesis of metabolites II, III, IV, VI and VII. The EI mass spectra of the bis-trimethylsilyl derivatives of boldenone and its metabolites display all intense molecular ions, M-15 ions and fragment ions originating from cleavage of the B-ring. The excreted metabolites can be separated in basic extractable labile conjugates and in stable conjugates. More than 95% of metabolites are excreted as stable conjugates.