Estrogen (E) and progesterone exert profound influence on development and reproduction. In vitro, steroid receptor coactivators (SRCs) are nuclear proteins that interact with DNA-bound steroid receptors to potentiate their transcriptional efficiency. We examined the effects of antisense oligonucleotides to SRC-1, SRC-2, and SRC-3 on female sexual behavior and steroid receptor-mediated transcription. Rat (r) SRC-1, rSRC-2, and rSRC-3 genes were cloned. Our results reveal a significant inhibitory effect by antisense (AS) to SRC-1 and SRC-2, but not SRC-3, on hormone-induced reproductive behavior. Importantly, sexual behavior was attenuated through estrogen receptor alpha (ERalpha)-dependent, rather than progesterone receptor (PR)-dependent, transcription, as E failed to induce the synthesis of PR content in the medial basal hypothalamus, and immunoreactive PR in the ventromedial nucleus were depleted in tissue from rSRC-1-AS- and rSRC-2-AS-treated, but not rSRC-3-AS-treated, rats primed with E. Consistent with interruption of ERalpha-induced transcription, high dose of E and epidermal growth factor alone failed to induce sexual behavior in females treated with either rSRC-1-AS or SRC-2-AS. Immunoreactive SRC-1 and SRC-2, but not SRC-3, proteins were abundant in the ventromedial nucleus, thus demonstrating that the biological activities of hypothalamic steroid receptors are selectively regulated by regional distribution of specific SRCs. As SRC-1 knockout mice have only a slight loss in reproductive function, the possibility that genetic adaptation occurs during development was tested. Mouse (m) SRC-1-AS suppressed lordosis in wild-type, but not SRC-1, knockout mice, whereas mSRC-2-AS suppressed behavior in both genotypes. mSRC-3-AS had no effect in either genotype, and SRC-3 knockout mice exhibited full receptivity. Collectively, the findings clearly implicate dual regulation of ERalpha-dependent function by SRC-1 and SRC-2 in the intact female brain. In the genetic, but not acute, absence of SRC-1, up-regulation of SRC-2 serves as a critical adaptive mechanism during female development.