Vitellogenin is a phospholipoglycoprotein precursor of egg yolk. In mature female fish, vitellogenin is synthesized and secreted by the liver in response to circulating estrogens. Vitellogenin is normally undetectable in the blood of male fish, but can be induced by exposure to compounds possessing estrogenic activity. Thus, the presence of vitellogenin in blood of male fish can serve as a useful biomarker for assessing previous exposure to estrogenic compounds. In the present study, we report identification and purification of vitellogenin in the mosquitofish (Gambusia affinis). Anti-vitellogenin immune serum was generated and used to develop an immunoblot assay for detection of vitellogenin. A combination of immunoblotting and densitometric scanning was used to assess the time- and dose-dependent effects of 17alpha-ethynylestradiol on vitellogenesis in male G. affinis. The results indicate that changes in the level of vitellogenin in mosquitofish blood can be reliably detected by the immunoblot assay, and that the mosquitofish may be a useful bioindicator organism for detecting estrogenic contamination of the aquatic environment.