Studies worldwide have demonstrated the occurrence of feminized male fish at sites impacted by human and animal wastes. A variety of chemicals could contribute to this phenomenon, but those receiving the greatest attention in terms of research and monitoring have been 17β-estradiol (β-E2) and 17α-ethinylestradiol, due both to their prevalence in the environment and strong estrogenic potency. A third steroid, estrone (E1), also can occur at high concentrations in surface waters but generally has been of lesser concern due to its relatively lower affinity for vertebrate estrogen receptors. In an initial experiment, male fathead minnow (Pimephales promelas) adults were exposed for 4-d to environmentally relevant levels of waterborne E1, which resulted in plasma β-E2 concentrations similar to those found in reproductively active females. In a second exposure we used 13C-labeled E1, together with liquid chromatography-tandem mass spectrometry, to demonstrate that elevated β-E2 measured in the plasma of the male fish was indeed derived from the external environment, most likely via a conversion catalyzed by one or more 17β-hydroxysteroid dehydrogenases. The results of our studies suggest that the potential impact of E1 as an environmental estrogen currently is underestimated.