To evaluate the potential role of endocrine disruption in the decline of pelagic fishes in the San Francisco Bay Delta of California, various surface water samples were collected, extracted, and found to elicit estrogenic activity in laboratory fish. Chemical analysis of the estrogenic samples indicated 2 pesticides (bifenthrin, diuron), 2 alkyphenols (AP), and mixtures of 2 types of alkyphenol polyethoxylates (APEOs). Evaluation of estrogenic activity was further characterized by in vitro bioassays using rainbow trout hepatocytes (Oncorhynchus mykiss) and in vivo studies with Japanese medaka (Oryzias latipes). In the in vitro bioassays, hepatocytes exposed to the pesticides alone or in combination with the AP/APEO mixtures at concentrations observed in surface waters failed to show estrogenic activity (induction of vitelloginin mRNA). In the in vivo bioassays, medaka exposed to individual pesticides or to AP/APEO alone did not have elevated VTG at ambient concentrations. However, when the pesticides were combined with AP/APEOs in the 7-day exposure a significant increase in VTG was observed. Exposure to a 5-fold higher concentration of the AP/APEO mixture alone also significantly induced VTG. In contrast to earlier studies with permethrin, biotransformation of bifenthrin to estrogenic metabolites was not observed in medaka liver microsomes and cytochrome P450 was not induced with AP/APEO treatment. These results showed that mixtures of pesticides with significantly different modes of action and AP/APEOs at environmentally relevant concentrations may be associated with estrogenic activity measured in water extracts and feral fish that have been shown to be in population decline in the San Francisco Bay Delta.