17β-Estradiol decreases R(+)8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding [an index of serotonin-1A (5-HT<sub>1A</sub>) receptor coupling] through the activation of estrogen receptors. We hypothesize that this occurs as a result of activation of protein kinase A (PKA) and/or protein kinase C (PKC) and phosphorylation of 5-HT<sub>1A</sub> receptors. Hippocampus from ovariectomized rats was incubated with 17β-estradiol in HEPES buffer (37°C). Cytosolic and membrane fractions were prepared to assess PKA and PKC activities, respectively. In separate experiments, membranes were prepared to measure R(+)8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding. 17β-Estradiol (50 n M) increased PKA and PKC activities approximately 2- to 3-fold. PKC activity was elevated at 10, 30 and 60 min, whereas PKA activity was increased at 10 and 30 min. The ability of 17β-estradiol to increase PKA and PKC was blocked by the estrogen receptor antagonist ICI 182,780 (1 µ M). A selective PKA inhibitor (KT 5720, 60 n M) blocked 17β-estradiol-stimulated PKA but not PKC activity. Conversely, the PKC inhibitor calphostin C (100 n M) blocked the increase in PKC activity produced by 17β-estradiol but not the PKA response. The protein kinase inhibitors individually blocked the effects of 17β-estradiol on R(+)8-OH-DPAT-stimulated [<sup>35</sup>S]GTPγS binding. By contrast, preincubation with the protein synthesis inhibitor cycloheximide (200 µ M) or the mitogen activated protein (MAP) kinase kinase inhibitor PD 98059 (50 µ M) was without effect. Incubation of hippocampus with 17β-estradiol (50 n M, 60 min) caused the phosphorylation of a protein consistent with the 5-HT<sub>1A</sub> receptor. These studies demonstrate that 17β-estradiol acts on estrogen receptors locally within the hippocampus through nongenomic mechanisms to activate PKA and PKC, phosphorylate 5-HT<sub>1A</sub> receptors and uncouple them from their G proteins.