Trenbolone acetate is a synthetic steroid that is extensively used in the United States as a growth promoter in beef cattle. The acetate is administered to livestock via slow-release implants; some is converted by the animal to 17-β-trenbolone, a relatively potent androgen receptor agonist in mammalian systems. Recent studies indicate that excreted 17-β-trenbolone is comparatively stable in animal waste, suggesting the potential for exposure to aquatic animals via direct discharge, runoff, or both. However, little is known concerning the toxicity of trenbolone to fish. Our goal was to assess the effects of 17-β-trenbolone on reproductive endocrinology of the fathead minnow (Pimephales promelas). An in vitro competitive binding study with the fathead minnow androgen receptor demonstrated that 17-β-trenbolone had a higher affinity for the receptor than that of the endogenous ligand, testosterone. Male and female fish were exposed for 21 d to nominal (target) concentrations of 17-β-trenbolone ranging from 0.005 to 50 μg/L. Fecundity of the fish was significantly reduced by exposure to measured test concentrations ≥ 0.027 μg/ L. The 17-β-trenbolone was clearly androgenic in vivo at these concentrations, as evidenced by the de novo production in females of dorsal (nuptial) tubercles, structures normally present only on the heads of mature males. Plasma steroid (testosterone and β-estradiol) and vitellogenin concentrations in the females all were significantly reduced by exposure to 17-β-trenbolone. The 17-β-trenbolone also altered reproductive physiology of male fathead minnows, albeit at concentrations much higher than those producing effects in females. Males exposed to 17-β-trenbolone at 41 μg/L (measured) exhibited decreased plasma concentrations of 11-ketotestosterone and increased concentrations of β-estradiol and vitellogenin. Overall, our studies indicate that 17-β-trenbolone is a potent androgen and reproductive toxicant in fish. Given the widespread use of trenbolone acetate as a growth promoter, and relative stability of its metabolites in animal wastes, further studies are warranted to assess potential ecological risk.