Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of exposure to (anti-)oestrogens and their mimics in fish [Sumpter, J.P., 1995. Feminized responses in fish to environmental estrogens. Toxicol. Lett. 82, 737-742; Arukwe, A., Goksøyr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. ]. In some instances, links have been drawn between high level induction of Vtg and adverse health effects in fish [Herman, R.L., Kincaide, H.L., 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture 72, 165-172; Schwaiger, J., Spieser, O.H., Bauer, C., Ferling, H., Mallow, U., Kalbfus, W., Negele, R.D., 2000. Chronic toxicity of nonylphenol and ethinyloestraiol: haematological and histopathological effects in juvenile common carp (Cyprinus carpio). Aquat. Toxicol. 51, 69-78]. The widespread use of Vtg as a biomarker has led to the development of a variety of assays to quantitatively measure Vtg concentrations in tissue samples from fish, and hence a need for a standardization of the performance criteria and validation of such assays [Goksøyr, A., Eidem, J.K., Kristiansen, S.I., Nilsen, B.M., 2003. On the need for a standardized set-up for validation studies of fish vitellogenin assays as an endpoint in endocrine disruptor testing and screening-a proposal. ]. One of the most popular test fish species for assessing chemical effects is the fathead minnow (Pimephales promelas), which is now used widely for studies into endocrine disruption [Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti)estrogenic substances. Environ. Toxicol. Chem. 21, 319-326; Hutchinson, T.H., Yokota, H., Hagino, S., Ozato, K., 2003. Development of fish tests for endocrine disruptors. Pure Appl. Chem. 75, 2343-2353]. This paper describes the development and validation of a new, homologous enzyme-linked immunosorbent assay (ELISA) for quantification of Vtg in this fish species.