We investigated the involvement of Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca(2+)-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca(2+)-independent activity. The autophosphorylation and appearance of Ca(2+)-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (alphaT286A kinase) or Asp (alphaT286D kinase) was examined. alphaT286A kinase was not converted to a Ca(2+)-independent form, and alphaT286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alphaT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing alphaT286D kinase had much longer neurites than Nb2a/alpha cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca(2+)-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.