Sexual differentiation in fish occurs after hatching during early life-stages and is believed to be a time when the gonad has a heightened sensitivity to disruption by chemicals that mimic hormones. In this study fathead minnows (Pimephales promelas) were exposed to an environmentally relevant concentration of ethinylestradiol (EE2) for short intervals in fish early life-stages and vitellogenic and gonadal responses were measured at 30 and 100 dph (sexual maturity), respectively. All EE2 exposure regimes induced vitellogenin (VTG) synthesis and disruption in duct development (a feminization) in males, with a window of enhanced sensitivity between 10 and 15 dph (where 60% of the males had feminized ducts). There was an altered pattern in sex cell development in males (inhibition of spermatogenesis) in the solvent controls (ethanol 0.1 ml/l) and all EE2 treatments when compared with the dilution water controls. Furthermore, fewer spermatozoa were observed in the testis of males exposed to EE2 from 15 to 20 dph and fertilized eggs (<24 h post-fertilization)-20 dph, compared with both the solvent and dilution water controls. These data show that short exposures of embryos/very early life-stage fathead minnows to an environmentally relevant concentration of EE2 lead to alterations in gonadal development that potentially have reproductive consequences and thus population level effects.