The ability of calcium/calmodulin-dependent protein kinase II (CaMKII) to become calcium independent after autophosphorylation makes this enzyme a temporal marker of neuronal activity. Here we show that the calcium-independent form of CaMKII has unique effects on larval viability, locomotion, and neuronal excitability in Drosophila. Expression of constitutively active T287D, but not calcium-dependent T287A, mutant CaMKII in Drosophila neurons resulted in decreased viability, behavioral defects, and failure of action potential propagation. The actions of T287D may be mediated, at least in part, by increased potassium conductances. Expression of T287D CaMKII also stimulated an increase in the number of boutons at the larval neuromuscular junction, but did not affect the mechanics of release. This study defines a role for autophosphorylation of CaMKII in the regulation of multiple neuronal functions including the intrinsic properties of neurons.