Multiple mechanisms mediate the effects of estrogen in the central nervous system, including signal transduction pathways such as protein kinase A, protein kinase C, and phosphatidylinositol 3‐kinase (PI3K) pathways. Previously we demonstrated that estrogen regulates a number of PI3K‐related genes in the hypothalamus, including the PI3K p55γ regulatory subunit. We hypothesized that PI3K activation is critical for the effects of estrogen and that the p55γ subunit may be more prevalent than the p85α regulatory subunit in the hypothalamus. Therefore, in the present study, we compared the mRNA distribution of the p55γ and p85α regulatory subunits by using in situ hybridization in guinea pig. Expression level of p55γ mRNA was greater than p85α in most hypothalamic nuclei. Twenty‐four hours of estrogen treatment increased p55γ mRNA expression in the paraventricular, suprachiasmatic, arcuate, and ventromedial nuclei, and little or no change was observed for p85α mRNA. Quantitative real‐time PCR confirmed the in situ hybridization results. Next, we investigated the general role of PI3K signaling in the estrogen‐mediated changes of arcuate proopiomelanocortin (POMC) neuronal excitability by using whole‐cell recording. One cellular mechanism by which estrogen increases neuronal excitability is to desensitize (uncouple) γ‐aminobutyric acid type B (GABA_B) receptors from their G‐protein‐gated inwardly rectifying K⁺ channels in hypothalamic neurons. We found that the PI3K inhibitors wortmannin and LY294002 significantly reduced the estrogen‐mediated GABA_B receptor desensitization in POMC arcuate neurons, suggesting that PI3K signaling is a critical downstream mediator of the estrogen‐mediated rapid effects. Collectively, these data suggest that the interplay between estrogen and PI3K occurs at multiple levels, including transcriptional and membrane‐initiated signaling events that ultimately lead to changes in homeostatic function. J. Comp. Neurol. 506:895–911, 2008. © 2007 Wiley‐Liss, Inc.