Previous studies of Eastern mosquitofish in contaminated Lake Apopka, Florida, have documented reduced sperm count and sexual behaviour in males but increased fecundity and liver weight in females, compared to nearby reference lakes. Liver weight can be an indicator of vitellogenin (Vtg) synthesis in fish, such as the mosquitofish. It was therefore hypothesized that estrogenic organochlorine pesticides, present at elevated concentrations in animals from Lake Apopka, could cause the reproductive disorders in males, as well as increase female fecundity. We initiated a test of this hypothesis by examining the relationship between 17beta-estradiol (E2) tissue concentrations, hepatic estrogen receptor alpha (ERalpha) and Vtg A, B and C gene expression and fecundity in sexually mature female Eastern mosquitofish from Lake Apopka and two reference lakes, Lake Woodruff and Lake Orange. We observed that female Eastern mosquitofish from one site in contaminated Lake Apopka produced fewer but bigger embryos than females from the other Lake Apopka site and two reference sites. However, female E2 concentrations and hepatic ERalpha and Vtg A, B and C gene expression showed no overall differences among the four sites, and it is therefore unlikely that the differences in fecundity were caused by estrogenic EDCs. In addition, we observed no induction of any of the three Vtg genes in male Eastern mosquitofish at the two Lake Apopka sites. Based on the well-documented high sensitivity of Vtg induction as a biomarker of xenoestrogen exposure, the evidence from the present study does not support the hypothesis that estrogenic EDCs are affecting reproduction in Eastern mosquitofish living in Lake Apopka. Our experimental design tested specifically for effects mediated via the ER, and e.g. antiandrogenic DDT metabolites might still be of importance for mosquitofish reproduction in Lake Apopka.