Reproductive functions are controlled by a finely tuned balance between estrogens and androgens. To further characterize the gonadal pathways leading to hormonal balance disruption by atrazine, vinclozolin, methoxychlor, and bisphenol A in rat, we investigated their effects in male and female young adult animals. Specifically, we assessed reproductive tract alterations, sex hormone balance in serum and gonads, tissue dosimetry, and mRNA expression. Remarkably, we observed different aromatase regulation profiles between animals with similar estrogen-to-androgen ratios but with different chemical treatments. For example, increased estrogen-to-androgen ratios in atrazine-treated females could be partly linked to aromatase upregulation, while in methoxychlor- and bisphenol A-treated females, peripheral mechanisms such as conjugation/deconjugation processes might be more likely to elevate estrogen levels. In vinclozolin-treated animals, the decreased estrogen-to-androgen ratios reported might be due to an increase of peripheral (adrenal) steroidogenesis. Thus, measurement of many endpoints is necessary for good risk assessment. Highlights Hormonal balance as a relevant endpoint for reproductive toxicology assessment. Characterization of gonadal pathways involved in endocrine disruption. Discrepancy between in vitro chemicals classification and in vivo reality. Several well chosen complementary endpoints are required for good risk assessment.