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Prenatal testosterone (T) treatment leads to polycystic ovarian morphology, enhanced follicular recruitment/depletion, and increased estradiol secretion. This study addresses whether expression of key ovarian genes and microRNA are altered by prenatal T excess and whether changes are mediated by androgenic or estrogenic actions of T. Pregnant Suffolk ewes were treated with T or T plus the androgen receptor antagonist, flutamide (T+F) from d 30 to 90 of gestation. Expression of steroidogenic enzymes, steroid/gonadotropin receptors, and key ovarian regulators were measured by RT-PCR using RNA obtained from fetal ovaries collected on d 65 [n = 4, 5, and 5 for T, T+F, and control groups, respectively] and d 90 (n = 5, 7, 4) of gestation. Additionally, fetal d 90 RNA were hybridized to multispecies microRNA microarrays. Prenatal T decreased (P < 0.05) Cyp11a1 expression (3.7-fold) in d 90 ovaries and increased Cyp19 (3.9-fold) and 5α-reductase (1.8-fold) expression in d 65 ovaries. Flutamide prevented the T-induced decrease in Cyp11a1 mRNA at d 90 but not the Cyp19 and 5α-reductase increase in d 65 ovaries. Cotreatment with T+F increased Cyp11a1 (3.0-fold) expression in d 65 ovaries, relative to control and T-treated ovaries. Prenatal T altered fetal ovarian microRNA expression, including miR-497 and miR-15b, members of the same family that have been implicated in insulin signaling. These studies demonstrate that maternal T treatment alters fetal ovarian steroidogenic gene and microRNA expression and implicate direct actions of estrogens in addition to androgens in the reprogramming of ovarian developmental trajectory leading up to adult reproductive pathologies.