The physical and chemical properties of purified catalytic and regulatory subunits of type II cAMP-dependent protein kinase from bovine brain, skeletal muscle, and cardiac muscle were compared. The catalytic subunits from all three sources were identical with respect to molecular weight (Mr = 40,000), amino acid composition, and isoelectric points. Furthermore, two-dimensional maps of their tryptic peptides were identical. The type II regulatory subunits from brain and skeletal muscle exhibited identical molecular weights by sodium dodecyl sulfate-gel electrophoresis and both undergo autophosphorylation; however, they differ somewhat in amino acid composition. Furthermore, two-dimensional tryptic peptide maps showed that 40% of the peptides differ and 60% co-migrate. Autoradiography of the peptide maps showed that the main phosphorylated peptide from these two sources also differ. The bovine brain type II regulatory subunit also differed from that of bovine cardiac muscle in the same manner. The molecular weights, amino acid composition, and tryptic peptide maps of the bovine skeletal and cardiac muscle regulatory subunits, however, were experimentally identical. These results suggest that type II protein kinases from bovine brain and muscle (skeletal or cardiac) represent distinct species which differ in their regulatory subunits but share a common catalytic subunit. The tryptic peptide map of the type I regulatory subunit from bovine skeletal muscle was very different from that of the two classes of type II regulatory subunit. There were, however, four polar peptides from neural and nonneural type II subunits as well as the type I regulatory subunit which co-migrated.