We have characterized chicken gizzard smooth muscle Ca2+/calmodulin-dependent protein kinase II (CaM-PKII) with particular focus on its autophosphorylation. The autophosphorylation of smooth muscle CaMPKII produced a partially constitutively active enzyme, as occurs with the alpha- and beta-isoforms of this enzyme, but the autophosphorylation kinetics were significantly slower. Phosphorylation during the initial rapid phase coincided with the production of constitutively active enzyme. The phosphorylation was on both serine and threonine residues, which is distinct from the brain enzyme where threonine phosphorylation is much faster and more prevalent than serine phosphorylation. The major autophosphorylation sites identified were different from the known autophosphorylation sites of the alpha- and beta-isoforms. During the initial autophosphorylation phase Ser-319, Ser-352 and a Thr residue within residues 345-368 were found to be phosphorylated. During the subsequent gradual phase two serine residues in the variable region and Ser-280 were phosphorylated, but Thr-286 and Thr-305, which are the known major autophosphorylation sites for the alpha- and beta-isoforms, were not detected as the major autophosphorylation sites of smooth muscle CaMPKII. By comparing the phosphopeptide sequence with the known sequences of various isoforms, we concluded that isoform gamma-b, which contains a unique insertion and two deletions at the C-terminal side of the calmodulin binding domain, is the dominant CaMPKII isoform in smooth muscle. The molecular mass of smooth muscle CaMPKII was estimated to be 240 kDa which would comprise four subunits, fewer than in the alpha- and beta-isoforms. The results show that smooth muscle CaMPKII is functionally distinct from the alpha- and beta-isoforms of this enzyme, which might be crucial for its physiological relevance.