The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.