Elevation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) in vascular smooth-muscle cells produces relaxation, but mechanisms distal to PKG activation are not well understood. Few PKG substrates have been described in smooth muscle that may mediate the action of PKG, including P240, P132 and phospholamban. None of them is a specific PKG substrate, raising the question of whether any specific PKG substrates possibly exist in vascular smooth muscle that may play roles in relaxation. In this study PKG substrates were detected in aortic smooth muscle by adding purified exogenous PKG and [γ-<sup>32</sup>P]-ATP. Very few PKG substrates were detectable in whole-tissue homogenates or detergent-solubilized fractions, due to the high basal activity of other protein kinases and the large numbers of other phospho-proteins. Heat or acid treatment of such fractions, to remove and endogenous protein kinase activity and achieve partial protein purification, revealed many potential PKG substrates. Of the 3 substrates identified previously, P240 and P132 were partly heat-stable. Thirty-one new PKG substrates were found: 14 in the initial heat-stable extract and 9 in the heat- and acid-soluble extract, whereas the others were revealed only after chromatography. All of the heat-stable PKG substrates were bound and salteluted from a DEAE-cellulose column in 2 major peaks called pool I and II. After sequential application to Q-Sepharose and S-Sepharose columns, 7 PKG substrates were found in pool I, in particular a group of 4 substrates of 40, 33, 28 and 22 kD virtually co-eluted through all 3 columns. The former 3 produced similar phosphopeptide maps, suggesting a relationship. All the new substrates from pool I were relatively specific for PKG because they were poorly phosphorylated with exogenous cAMP-dependent protein kinase and not with Ca<sup>2+</sup>/phospholipid-depen-dent protein kinase. Further chromatography of the proteins in pool II resulted in an extensive purification of P132 as well as a group of 4 PKG substrates of 33-30 kD. Phosphopeptide mapping of the 132-kD protein revealed a close homology to the 132-kD PKG substrate previously described in rat aortic smooth muscle. These data demonstrate the presence of multiple substrates for PKG in aortic smooth-muscle tissue.