Among the three major vascular layers (the intima-inner, the media-middle smooth muscle, and the adventitia-outer connective tissue) over 90% of the total protein kinase activity was observed in the middle layer. Of various subcellular fractions of the vascular smooth muscle, the 105,000 g supernatant (cytosol fraction) showed the highest specific activity and represented more than two thirds of the total kinase present in this layer. DEAE-cellulose chromatography of the soluble enzyme revealed the existence of two major forms of cyclic AMP-dependent protein kinase, type I and type II, of which 60% of the total enzymatic activity was found in type II. A divalent cation was found to be essential for their phosphotransferase activity. Only Mg<sup>2+</sup> and Co<sup>2+</sup>, but not Zn<sup>2+</sup>, Mn<sup>2+</sup> or Ca<sup>2+</sup> could satisfy the cation requirement. The phosphorylated substrate had the characteristics of a protein with a phosphoester bond.