In this study, we characterized the effects of flutamide, a model mammalian androgen receptor (AR) antagonist, on endocrine function in the fathead minnow (Pimephales promelas), a small fish species that is widely used for testing endocrine-disrupting chemicals (EDCs). Binding assays with whole cells transiently transfected with cloned fathead minnow AR indicated that flutamide binds competitively to the receptor. However, as is true in mammalian systems, a 2-hydroxylated metabolite of flutamide binds to the AR with a much higher affinity than the parent chemical. Mixture experiments with flutamide and the androgen 17beta-trenbolone demonstrated that the anti-androgen effectively blocked trenbolone-induced masculinization (nuptial tubercle production) of female fathead minnows, indicating antagonism of an AR receptor-mediated response in vivo. Conversely, reductions in vitellogenin in trenbolone-exposed females were not blocked by flutamide, suggesting that the vitellogenin response is not directly mediated through the AR. The results of these studies provide data demonstrating the validity of using the fathead minnow as a model species for detecting EDCs that exert toxicity through interactions with the AR.