The ubiquitous presence of nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) compounds in aquatic environments adjacent to wastewater treatment plants (WWTP) warrants an assessment of the endocrine disrupting potential of these complex mixtures on aquatic vertebrates. In this study, fathead minnow larvae were exposed for 64 days to a mixture of NPE/OPE, which closely models the NPE/OPE composition of a major metropolitan WWTP effluent. Target exposure concentrations included a total NPE/OPE mixture load of 200% of the WWTP effluent concentration (148microg/L), 100% of the WWTP effluent concentration (74microg/L) and 50% of the WWTP effluent concentration (38microg/L). The NPE/OPE mixture contained 0.2% 4-t-octylphenol, 2.8% 4-nonylphenol, 5.1% 4-nonylphenolmonoethoxylate, 9.3% 4-nonylphenoldiethoxylate, 0.9% 4-t-octylphenolmonoethoxylate, 3.1% 4-t-octylphenoldiethoxylate, 33.8% 4-nonylphenolmonoethoxycarboxylate, and 44.8% 4-nonylphenoldiethoxycarboxylate. An additional exposure of 5microg/L 4-nonylphenol (nominal) was conducted. The exposure utilized a flow-through system supplied by ground water and designed to deliver consistent concentrations of applied chemicals. Following exposure, larvae were raised to maturity. Upon sexual maturation, exposed male fish were allowed to compete with control males in a competitive spawning assay. Nest holding ability of control and exposed fish was carefully monitored for 7 days. All male fish were then sacrificed and analyzed for plasma vitellogenin, developmental changes in gonadal tissues, alterations in the development of secondary sexual characters, morphometric changes, and changes to reproductive behavior. When exposed to the 200% NPE/OPE treatment most larvae died within the first 4 weeks of exposure. Both the 100% and 50% NPE/OPE exposures caused a significant decrease in reproductive behavior, as indicated by an inability of many of the previously exposed males to acquire and hold a nest site required for reproduction. In contrast, the 5microg/L 4-nonylphenol exposure resulted in significantly enhanced reproductive behavior compared to that of control males and a majority of the nesting sites were held by previously exposed males. No significant change in the development of gonadal tissues was observed. The 100% NPE/OPE exposure resulted in a significant reduction in the gonadal somatic index and in the prominence of secondary sexual characteristics of exposed larvae. This study indicates that NPE/OPE mixtures have an effect on the reproductive competence of previously exposed male fathead minnows. In addition, 4-nonylphenol concentrations utilized in all exposures were below regulatory guidelines, suggesting that evaluation of 4-nonylphenol alone may not be sufficient for identifying potentially adverse effects of this suite of compounds usually found as mixtures in the aquatic environment.