Previously our laboratory has shown that 17β-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT<sub>1A</sub> receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17β-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT<sub>1A</sub> receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding assays. 17β-Estradiol (10<sup>–6</sup> M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC<sub>50</sub> of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17β-estradiol for 1 h, the calculated EC<sub>50</sub> was approximately 2.5 × 10<sup>–8</sup> M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT<sub>1A</sub> receptor function while the less potent estrogens 17α-estradiol and estriol were inactive at 5 × 10<sup>–8</sup> M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17β-estradiol on 5-HT<sub>1A</sub> receptor function with an apparent K<sub>B</sub> of approximately 10<sup>–9</sup> M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT<sub>1A</sub> receptor function.