Abstract: Ca2+/calmodulin-dependent protein kinase II(CaMK-II) has been linked to the induction of differentiation in preneuronalcells. In these cells, δ isozymes represent the majority of CaMK-IIsexpressed and are activated by differentiation stimuli. To determine whetherδ CaMK-IIs are causative or coincident with in vitro differentiation, weoverexpressed wild-type, constitutively active, and C-terminal domains ofδ and γ CaMK-II isozymes in mouse P19 and NIH/3T3 cells usinghigh-efficiency transfections. At 1-2 days after transfection, onlyconstitutively active δ CaMK-II isozymes induced branched cellularextensions in both cell types. In P19 cells, retinoic acid induced neuriteextensions after 3-4 days; these extensions were coincident with a fourfoldincrease in endogenous CaMK-II activity. Extensions induced by both retinoicacid and δ CaMK-IIs contained class III β-tubulin in adiscontinuous or beaded pattern. C-terminal CaMK-II constructs disrupted theability of endogenous CaMK-II to autophosphorylate and blocked retinoicacid-induced differentiation. δ CaMK-II was found along extensions,whereas γ CaMK-II exhibited a more diffuse, cytosolic localization.These data not only support an extranuclear role for CaMK-II in promotingneurite outgrowth, but also demonstrate CaMK-II isozyme specificity in theseearly steps of neuronal differentiation.