Ca2+/CaM-dependent multifunctional protein kinase isoenzymes from brain, skeletal muscle and liver were compared by their phosphorylation of a number of protein substrates. Under the conditions of assay, the three isoenzymes demonstrated rapid phosphorylation of synapsin I and glycogen synthase. In contrast, rates of phosphorylation of pyruvate kinase and phenylalanine hydroxylase were almost two orders of magnitude slower. Differences in phosphorylation specifically of the latter two substrates was also observed among the three protein kinases. Phosphorylation by Ca2+/CaM-dependent protein kinases was contrasted with cAMP-dependent protein kinase, which phosphorylates these proteins in vitro and in vivo. The potential role of Ca2+/CaM-dependent multifunctional protein kinases in the Ca2+-dependent phosphorylation of these substrates is discussed.