The Santa Ana River (SAR) in southern California is impacted by effluents from wastewater treatment plants (WWTP), which are sources of organic wastewater compounds (OWCs) and urban runoff. The Santa Ana River is one of only three river basins supporting native populations of the federally listed Santa Ana sucker (Catostomus santaanae) at the time the fish was included on the list 2000. In 2004 and 2005, a U.S. Geological Survey and U.S. Fish and Wildlife Service study was undertaken to determine if the threatened Santa Ana sucker was potentially exposed to OWCs and endocrine disrupting compounds (EDCs) in the SAR by using the western mosquitofish (Gambusia affinis) as a surrogate fish model. Four Santa Ana River sites were chosen along a gradient of proximity to WWTP effluents: (1) a point source of tertiary treated wastewater effluent (TTWE), (2) Rialto Drain (just below a WWTP), (3) Prado Dam (11 kilometers [km] below WWTPs), and (4) Sunnyslope Creek (no WWTP but having urban runoff influence). A reference site having no WWTPs or urban runoff, Thousand Palms, was also sampled. Chemical analyses of passive sampler extracts results showed that 15 OWCs and EDCs were detected in water from the Santa Ana River sites. Many of these compounds contributed to activity from an estrogenic in-vitro assay that showed a significant potential for impacting endocrine and reproductive systems compared to the 25 organochlorine compounds detected in aquatic biota. The site showing compounds having highest influence on sex steroid hormone activities was the point source for TTWE. Sex steroid hormone levels, secondary sex characteristics, organosomatic indices, and sperm quality parameters indicated impairment of endocrine and reproductive function of male western mosquitofish in the Santa Ana River. Exposure to EDCs and consequent impairment in mosquitofish followed the gradient of proximity to WWTP effluents, where the most significant effects were found at TTWE point source and Rialto Drain, followed by Prado Dam and Sunnyslope Creek. Each of these sites is suitable habitat for the Santa Ana sucker, especially Sunnyslope Creek and Rialto Drain where juveniles reside. Various OWCs and EDCs were detected at each Santa Ana River site, although one specific compound or group of compounds could not be singled out as a causative factor. Di (2-ethylhexyl) phthalate was strongly negatively correlated with testosterone in male mosquitofish. One group of potent environmental estrogens that likely contributed to endocrine and reproductive impairment are the natural and synthetic estrogen hormones, especially ethinyl estradiol; however, this compound was not targeted in these investigations. The multiple lines of evidence for impaired reproductive and endocrine function in western mosquitofish due to OWCs and EDCs from the Santa Ana River can be used to identify potential problems for the Santa Ana sucker inhabiting the same and nearby sites.