Many chemicals that are not structurally related to estrogen have estrogen-like activity. In this study, we tried to apply a quail embryo culture system for assessing the in vivo effects of such chemicals on the development of quail embryos. beta-Estradiol induced feminization of the gonads of genetically male embryos, which was confirmed by the increase in the size of the left gonad and female-specific aromatase expression, while male-specific SOX9 expression was not affected. Nonylphenol, which has a weak estrogenic activity, reduced the viability and body weight of embryos. Simultaneously, several genetically male embryos were feminized in terms of gonadal size and aromatase expression. These results indicate that the avian embryo culture system was useful for evaluating endocrine disrupters.