A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose Cl 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca2+/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase VI.