To examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the alpha-subunit of CaMKII (alpha CaMKII) equivalent to mammalian neurons. The expression of the transfected alpha CaMKII gene or the endogenous beta CaMKII gene was monitored by RNase protection assays, and alpha CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of alpha CaMKII mRNA and alpha CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from alpha CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing alpha CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the alpha CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of alpha CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that alpha CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.