A method to determine twelve anabolic hormones (diethylstilbestrol, hexestrol, dienestrol, 17 beta-estradiol, 19-nortestosterone, testosterone, 1-dehydrotestosterone, 17 alpha-methyltestosterone, progesterone, estrone, 17 alpha-ethynilestradiol, and trenbolone) is presented. Urine samples were extracted with octadecylsilica columns and clean-up was performed in two steps with basic alumina and silica solid-phase extraction cartridges. The extracts obtained were derivatized with heptafluorobutyric anhydride and analyzed by GC-MS. Stability of derivatives was good and compounds having keto groups produced enol derivatives that were stable also. SIM mode was applied to increase the sensitivity and, when possible, the higher m/z ions were selected to improve identification. Repeatability of the chromatographic analysis was evaluated on the basis of area repeatability, and the coefficient of variation obtained was lower than 13%. Absolute recoveries were in the range 35-60% (dehydrotestosterone and estrone < 20%) with coefficients of variation between 14 and 37% for the whole procedure. [2H3]Testosterone and [2H8]diethylstilbestrol were evaluated to improve quantitative data. The recovery of [2H3]testosterone was found to be equal to or slightly higher than that of the other hormones, but the recovery of [2H8]diethylstilbestrol was lower than any other. [2H3]Testosterone was the most suitable for use as an internal standard, as its addition at the beginning of analytical procedure, corrected recovery results and greatly improved precision. Corrected recoveries from urine ranged from 72-110%, and coefficients of variation ranged from 6-15%, except for testosterone which yielded slightly higher values. The limit of detection was 0.5 ng/ml for all the compounds studied.