Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase consisting of two isoforms, alpha and beta. The activities of GSK-3 are regulated negatively by serine phosphorylation but positively by tyrosine phosphorylation. GSK-3 inactivation has been proposed as a mechanism to promote neuronal survival. We used GSK-3 isoform-specific small interfering RNAs, dominant-negative mutants, or pharmacological inhibitors to search for functions of the two GSK-3 isoforms in regulating neuronal survival in cultured cortical neurons in response to glutamate insult or during neuronal maturation/aging. Surprisingly, RNA interference-induced depletion of either isoform was sufficient to block glutamate-induced excitotoxicity, and the resulting neuroprotection was associated with enhanced N-terminal serine phosphorylation in both GSK-3 isoforms. However, GSK-3beta depletion was more effective than GSK-3alpha depletion in suppressing spontaneous neuronal death in extended culture. This phenomenon is likely due to selective and robust inhibition of GSK-3beta activation resulting from GSK-3beta Ser(9) dephosphorylation during the course of spontaneous neuronal death. GSK-3alpha silencing resulted in reduced tyrosine phosphorylation of GSK-3beta, suggesting that tyrosine phosphorylation is also a critical autoregulatory event. Interestingly, GSK-3 inhibitors caused a rapid and long-lasting increase in GSK-3alpha Ser(21) phosphorylation levels, followed by a delayed increase in GSK-3beta Ser(9) phosphorylation and a decrease in GSK-3alpha Tyr(279) and GSK-3beta Tyr(216) phosphorylation, thus implying additional levels of GSK-3 autoregulation. Taken together, our results underscore important similarities and dissimilarities of GSK-3alpha and GSK-3beta in the roles of cell survival as well as their distinct modes of regulation. The development of GSK-3 isoform-specific inhibitors seems to be warranted for treating GSK-3-mediated pathology.