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 III) from rat pancreas. The physical properties and substrate specificity of CaM kinase III were distinct from those of all known CaM-dependent protein kinases. Only CaM kinase III 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 III revealed that the enzyme had a similar widespread tissue distribution. These results demonstrate the existence of a fifth CaM-dependent protein phosphorylation system present in high levels in animal cells.