Analytical methodology is described for determining residues of the synthetic anabolic steroid trenbolone acetate (TBA) and its hydrolysis product trenbolone (TBOH) in admixture in animal chow, human urine, and wastewater. Benzene extracts of the substrates are subjected to liquid-liquid partitioning, further cleanup on a column of silica gel, and direct analysis by high pressure liquid chromotography or derivatization with pentafluoropropionic anhydride and analysis by electron capture gas chromatography. Satisfactory recoveries were obtained with both compounds from all three substrates. Residue levels of TBA and TBOH as low as 0.32 and 0.04 ppm, respectively, could be detected in chow; about 0.6 ppb of each compound could be detected in urine and wastewater. Thin layer chromatographic behavior of the two compounds in 7 solvent systems and other ancillary analytical data are also presented.