To investigate the physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the alpha subunit of mouse CaM kinase II (CaM kinase II alpha) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase II alpha gene. The expression of CaM kinase II alpha was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the gamma and delta subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase II alpha through autophosphorylation and generation of the Ca2+/calmodulin-dependent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase II alpha. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.