Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of alpha and beta/beta' subunits of molecular weights 46,000 and 55,000/52,000, respectively. The ratio of alpha to beta/beta' subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The Km value of ATP was 19 +/- 1.3 microM, and the K0.5 values of calcium and calmodulin were 4.4 +/- 0.5 and 3.0 +/- 0.5 microM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the alpha-subunit but apparently not of the beta/beta' subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II alpha subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both alpha and beta subunits of hen brain CaM-kinase II cross-reacted with only the alpha subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II in several properties. Immunocross-reactivity suggested that there was similarity in the alpha but not the beta/beta' subunits of the hen brain enzyme and the brain enzyme of other species.