The estrogen receptor (ER) subtypes, ERalpha and ERbeta, modulate numerous signaling cascades in the brain to result in a variety of cell fates including neuronal differentiation. We report here that 17beta-estradiol (E2) rapidly stimulates the autophosphorylation of alpha-Ca(2+)/calmodulin-dependent kinase II (alphaCaMKII) in immortalized NLT GnRH neurons, primary hippocampal neurons, and Cos7 cells co-transfected with ERalpha and alphaCaMKII. The E2-induced alphaCaMKII autophosphorylation is ERalpha- and Ca(2+)/calmodulin (CaM)-dependent. Interestingly, the hormone-dependent association of ERalpha with alphaCaMKII attenuates the positive effect of E2 on alphaCaMKII autophosphorylation, suggesting that ERalpha plays a complex role in modulating alphaCaMKII activity and may function to fine-tune alphaCaMKII-triggered signaling events. However, it appears as though the activating signal of E2 dominates the negative effect of ER since there is a clear, positive downstream response to E2-activated alphaCaMKII; pharmacological inhibitors and RNAi technology show that targets of ERalpha-mediated alphaCaMKII signaling include extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding protein (CREB), and microtubule associated protein 2 (MAP2). These findings suggest a novel model for the modulation of alphaCaMKII signaling by ERalpha, which provides a molecular link as to how E2 might influence brain function.