A major substrate, Mr 100,00 (100 kDa), for a Ca2+/calmodulin (CaM)-dependent protein kinase found in many mammalian tissues has been purified from rat pancreas. The purified substrate was used to identify and partially purify a CaM-dependent protein kinase (CaM kinase Il) from rat pancreas. The physical properties and substrate specificity of CaM kinase HI were distinct from those of all known CaMdependent protein kinases. Only CaM kinase m was able to phosphorylate the 100-kDa protein; synapsin I, phosphorylase b, myosin light chain, and histone were poor substrates for this enzyme. Polyclonal antibodies, raised against the purified 100-kDa protein, recognized the protein in a variety of mammalian tissues and cell lines. Immunoassay revealed that the 100-kDa protein made up 0.3-1.7% of the total cytosolic protein in these samples. Analysis of CaM kinase m revealed that the enzyme had a similar widespread tissue distribution.