Estrogenic activity in fish has primarily been evaluated using vitellogenin expression in male and juvenile animals. Although the response has been widespread, the relevance of the response to higher-level adverse effects, particularly in the field is less than clear. Previous evaluations of vitellogenin within flatfish species collected near the Orange County Sanitation District (OCSD) outfall and a reference station 7.7 km downcurrent indicated bioavailable estrogens within demersal flatfish populations. In order to evaluate the persistence of estrogenic activity and relationships to reproduction and development, sediments were sampled in 2002, and fish were sampled in the winter and summer of 2003, and the winter and summer of 2004 from the same locations. Concentrations of estrogens and alkylphenol ethoxylates and alkylphenols were determined in the sediments. Vitellogenin, gonadal somatic indices, serum estradiol, and sperm DNA damage were measured in the fish. In addition gender ratios were measured in English sole (Pleuronectes vetulus) and hornyhead turbot (Pleuronichthys verticalis) collected in the summers of 2001 through 2004, and 1988 through 2004, respectively. Measurable concentrations of estradiol were observed in all sediment extracts including the proposed reference sediment and ranged from 0.16 to 0.45 ng/g. Alkylphenols and Alkylphenol ethoxylates were observed in all sediment samples but were highest near the OCSD outfall where concentrations of nonylphenol were 3200 ng/g. Although vitellogenin levels were elevated at the outfall compared to the reference station, the elevation was not significant. Rather than an expected feminization of populations, there was either no trend or a significant trend toward masculinization, as was observed in hornyhead turbot at the OCSD outfall (64% males at outfall versus 51% males at reference station). Male gonadal somatic indices were significantly higher at the outfall for English sole during the summer 2003 and 2004 sampling periods, and in hornyhead turbot males during the winter of 2004. Serum levels of estradiol were significantly decreased in female English sole from the outfall location during the winter of 2003. These results indicate possibly stronger androgenic activity than estrogenic activity on flatfish residing near the outfall.