Female sheep exposed to excess testosterone (T) during prenatal life, display an array of symptoms similar to those observed in women with polycystic ovarian syndrome (PCOS). Specifically, prenatal T-treated ewes display masculinized sexual behavior and increased food-related reward seeking behavior. A neural substrate critical for these goal-directed behaviors is the dopaminergic system in the ventral tegmental area. We have recently shown that in adult ewes dopamine expression in the VTA is increased by prenatal T exposure. In the current study, we tested the hypothesis that alterations of the VTA dopamine system by prenatal-T is caused via activation of androgen receptors, by testing if prenatal co-treatment of T with androgen receptor blocker flutamide attenuates the effects of prenatal T. Ewes were exposed prenatally to 1) T (twice weekly 2 ml intramuscular injections of 100 mg testosterone propionate; ~1.2 mg/kg ) during gestational days 30-90 of the 147 gestation period (T; n=5), 2) T plus flutamide, an androgen antagonist (TF; additional daily subcutaneous injections of flutamide; 15 mg/kg; n=11), 3) flutamide alone (CF; n=7), or vehicle (Control, C; n=7). At two years of age, all ewes were ovariectomized and administered steroid hormones to mimic late follicular phase. Brains were harvested and VTA DA expression was analyzed using immunohistochemistry for tyrosine hydroxylase (TH). In addition, co-expression of TH with androgen receptors (AR) was determined. Quantitative analysis of TH and AR expression confirmed the increase in TH expression and also showed increased AR after prenatal T treatment, specifically in TH neurons. Moreover, these effects were prevented by prenatal co-treatment with flutamide, as TH and AR expression in TF females were similar to control females, and significantly lower compared to prenatal T females. These results indicate that prenatal T organizes the VTA dopamine system via actions on androgen receptors.