The autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase was stimulated by the acidic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol. Other phospholipids (phosphatidylethanolamine, phosphatidylcholine, sphingomyelin), acidic compounds (dextran sulfate, polyglutamic acid, chondroitin sulfate, hyaluronic acid) and calciumcalmodulin were essentially inactive. Sodium dodecyl sulfate also stimulated the catalytic subunit autophosphorylation, but other detergents (Triton X-100 and deoxycholic acid) did not. The combination of phosphatidic acid and sodium dodecyl sulfate was as effective as each agent alone, suggesting similar stimulation mechanisms. The data suggest that acidic membrane phospholipids might have a role in regulating the autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.