It has been well established that the NGF-mediated survival of sympathetic neurons in culture occurs through the phosphatidylinositol (PI) 3-kinase/Akt-dependent pathway. In contrast, the mechanism by which membrane depolarization promotes neuronal survival independently of NGF remains unresolved. Here we show that LY294002, a specific inhibitor of PI 3-kinase, induced cell death of sympathetic neurons under depolarizing conditions with elevated K + (IC 50=∼30 μM). Interestingly, lower concentrations of this agent (≤10 μM) were sufficient to suppress Akt phosphorylation at Ser-473, a putative downstream target of PI 3-kinase, under these conditions. We also show that KN-62, a specific inhibitor of Ca 2+/calmodulin-dependent protein kinase II (CaMKII) suppressed depolarization-mediated survival in a does-dependent manner (IC 50=∼2 μM) that paralleled attenuation of sustained levels of intracellular Ca 2+ evoked by depolarization. This IC 50 value is greater than that for CaMKII (∼0.8 μM). These findings led us to hypothesize that depolarization-mediated survival occurs through both the PI 3-kinase/Akt and the CaMKII pathways. Indeed, combined treatment with LY294002 (25 μM) and KN-62 (0.5 μM) dramatically abolished depolarization-mediated survival, whereas each alone did not significantly attenuate it. Under these conditions, KN-62 neither impaired sustained levels of intracellular Ca 2+, nor inhibited the phosphorylation of Akt. It is thus likely that PI 3-kinase and CaMKII independently promote the membrane depolarization-mediated survival of sympathetic neurons in culture.