In human erythrocytes large portions of both adenosine 3′, 5′-monophosphate (cyclic AMP**)dependent protein kinase [EC 2. 7. 1. 37] and its substrate proteins are associated with membranes. On fractionation of the membrane multiple species of substrate proteins have been distinguished: one is tightly bound to the membrane-structure and others are extractable with salt solutions of higher ionic strength. The membrane-bound substrate is heat-stable. After lipid extraction this protein forms highly insoluble polymeric aggregates, but is fully active as substrate for the protein kinase. Before lipid extraction, however, the membrane-bound substrate may be solubilized with lithium diiodosalicylate, but a minimum molecular weight of about 65,000 has been obtained in the presence of sodium dodecyl sulfate. The soluble substrate proteins extracted from the membrane show relatively small molecular weights ranging from 12,000 up to about 30,000. The protein kinase associated with membranes is almost completely extracted as a soluble form with salt solutions. This kinase shows essentially similar kinetic and catalytic properties to many cyclic AMP-dependent protein kinases distributed in soluble fraction of various mammalian tissues.