Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) gamma-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II gammaB- and gammaC-subunits and a novel gamma-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the gammaG-subunit. A clone predicted to encode a 62. 2-kDa gamma-subunit, designated as gammaE, was isolated with a variable domain structure similar to a gammaB-subunit but with a 114-nucleotide insertion in the conserved "association" domain of CaM kinase II subunits. A full-length gammaE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of gammaB-, gammaC-, or gammaG-subunits. Expression of gammaE and related gamma-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of gamma-subunit expression: vascular smooth muscle, gammaB > gammaC > gammaE,G; heart, gammaB > gammaE,C > gammaG; brain, gammaE and related subunits >> gammaA,B,C,G.