Abstract: Recent studies have detected estrogenic compounds in surface waters in North America and Europe. Furthermore, the presence of estrogenic compounds in surface waters has been attributed, in some cases, to the discharge of wastewater treatment plant (WWTP) effluent. The primary objective of the current study was to determine if WWTP effluent contributes estrogens to the surface waters of Nebraska. A second objective of this study was to determine if estrogens were found in concentrations sufficient enough to manifest feminizing effects on fish. These objectives were satisfied by deploying polar organic chemical integrative samplers (POCIS) and caged fathead minnows at eight field sites. Deployment sites included: three reference sites (Pawnee Creek, the Little Blue River, and the Middle Loup River), two sites upstream of the WWTPs at Grand Island and Columbus, and three sites downstream of the WWTPs at Grand Island, Columbus, and Hastings. Following the seven day deployments, POCIS extracts were analyzed for estrone, 17β-estradiol, estriol and 17α-ethinylestradiol using liquid chromatography tandem mass spectrometry (LC/MS/MS). 17β-estradiol was detected in POCIS from six of the eight field sites with the greatest quantities recovered in POCIS deployed downstream from the Grand Island and Hastings WWTPs. Estrone was detected only in the POCIS deployed downstream from the Grand Island and Hastings WWTPs. Estrogenic effects were detected in caged minnows analyzed for the hepatic mRNA expression of two estrogen-responsive genes, vitellogenin (vg1) and estrogen receptor α (ERα). Fish deployed at the site where the greatest quantities of estrogens were recovered (Hastings) had significantly higher expression of both vg1 and ERα than fish deployed at any of the other sites. These results confirm that WWTP effluent contributes biologically significant levels of estrogens to Nebraska surface waters.