To contribute to the development and evaluation of a practical and cost-effective in vivo testing system for endocrine disruption (specifically environmental estrogens), the effects of 17alpha-ethinylestradiol (EE2) were assessed in a gonadal recrudescence assay with the fathead minnow (Pimephales promelas). Mature male and female fathead minnows were kept first at 15 degrees C on a 8 h light/16 h dark regime and then transferred to 25 degrees C and a 16 h light/8 h dark regime to induce gonadal recrudescence. They were then exposed to various nominal concentrations of the synthetic estrogen EE2 (0, 0.1, 1, 3, 10, 100 ng/L). After 3 weeks of chemical exposure, effects on plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI), and condition factor were assessed. Additionally, the effects on liver and gonad tissue morphology were investigated by means of light (LM) and electron microscopy (EM). Reproductive output (egg production) and fertilization rate were measured during a subsequent 3-week period in breeding adults maintained in clean water. Exposure to EE2 resulted in a significant decrease in GSI, condition factor, and number of batches of eggs and their fertilization rate at EE2 exposure concentrations between 10 and 100 ng/L. A reduction in the extent of parenchymatic areas in ovaries and ultrastructural changes in the livers of females could be detected at EE2 concentrations > or =3 ng/L. The lowest observed effective concentration (LOEC) of EE2 for plasma VTG induction in both sexes and for ultrastructural changes in the testes and livers was 1 ng/L. A significant increase in the mean number of eggs spawned per pair occurred at EE2 exposure doses of 0.1 and 1 ng/L. However, at higher EE2 concentrations, a dose-dependent decrease in the mean number of eggs per pair was apparent. Therefore, the LOEC for a biological effect of EE2 in the fathead minnow using the selected endpoints in the recrudescence assay was 1 ng/L for biomarkers such as plasma VTG and number of tubercles, and 0.1 ng/L for an increased number of eggs spawned per pair.