Testicular steroids during midgestation sexually differentiate the steroid feedback mechanisms controlling GnRH secretion in sheep. To date, the actions of the estrogenic metabolites in programming neuroendocrine function have been difficult to study because exogenous estrogens disrupt maternal uterine function. We developed an approach to study the prenatal actions of estrogens by co-administering testosterone (T) and the androgen receptor antagonist flutamide, and tested the hypothesis that prenatal androgens program estradiol inhibitory feedback control of GnRH secretion to defeminize (advance) the timing of the pubertal rise in LH. Pregnant sheep were either untreated or treated with T, dihydrotestosterone (DHT, a non-aromatizable androgen), or T+flutamide from Days 30–90 of gestation. To study the postnatal response to steroid negative feedback, lambs were gonadectomized and estradiol-replaced, and concentrations of LH were monitored in twice weekly blood samples. While T and DHT produced penile and scrotal development in females, the external genitalia of T+flutamide offspring remained phenotypically female, regardless of genetic sex. Untreated females and females and males treated with T+flutamide exhibited a pubertal rise in circulating LH at 26.4 ± 0.5, 26.0 ± 0.7, and 22.4 ± 1.6 weeks of age, respectively. In females exposed to prenatal androgens, the LH rise was advanced (T:12.0 ± 2.6 weeks; DHT:15.0 ± 2.6 weeks). These results demonstrate the usefulness of combining testosterone and antiandrogen treatments as an approach to increasing prenatal exposure to estradiol. Importantly, the findings support our hypothesis that prenatal androgens program sensitivity to the negative feedback actions of estradiol and the timing of neuroendocrine puberty.