Three distinct isozymes of a type II calcium/calmodulin-dependent protein kinase (CaM kinase II) from rat forebrain cytosol were separated using S-Sepharose cation-exchange resin. About 90% of the applied kinase activity was recovered in three protein peaks. Each isozymic form of the kinase was purified 200-300 fold by chromatography on S-Sepharose, calmodulin-affinity and gel filtration resins. All 3 forms of CaM kinase II had apparent molecular masses of 650-700 kDa, but contained variable proportions of 50 kDa and 58-60 kDa subunits. The molar ratios of the 50 kDa/58-60 kDa kinase subunits in each holoenzyme were determined by protein staining and [125I]calmodulin-binding studies and were approximately: 6/1, 3/1 and 1/1. The isozyme containing a 3/1 ratio of subunits corresponds to the predominant form of CaM kinase II in forebrain representing 70-80% of the total activity in cytosol, whereas the other forms each represent 5-10% of the total cytosolic activity. The substrate specificities and time courses of substrate phosphorylation for the isozymes were comparable. These studies provide a basis to examine regional, subcellular, and developmental differences in the isozymic forms of CaM kinase II which may subserve different neuronal functions.