17β-Estradiol can potentiate kainate-induced currents in isolated hippocampal CA1 neurons. The action of estrogen was rapid in onset, steroid and stereospecific, and reversible. The potentiation could be mimicked by 8-bromo-cAMP, an activator of protein kinase A. As the hippocampus expresses both isoforms of the intracellular estrogen receptor (ERα and ERβ), the role of ERs in the rapid action of 17β-estradiol remains elusive. Here we report that the rapid action of 17β-estradiol is independent from the classical ER activation in the modulation of membrane excitability. Under whole cell voltage clamp recording configuration, 17β-estradiol-induced potentiation was observed in both wild-type and the ERα gene knockout mice. The perfusion or incubation of ICI 182,780, which blocks both ERα and ERβ, did not affect estrogen potentiation in either group. Further study showed that adenosine 3′,5′-cyclic-monophosphothioate Rp-isomer, a specific inhibitor of protein kinase A, completely blocked the potentiation observed with the application of 17β-estradiol in ERα gene knockout mice. Our results provide evidence that a distinct estrogen-binding site exists, which appears to be coupled to α-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid/kainate receptors by a cAMP-dependent phosphorylation process.