In intact blood vessels, many vasodilators act by stimulating the release from endothelium of factor(s) that relax vascular smooth muscle and stimulate increases in cGMP. To investigate how endothelium regulates cGMP production in vascular smooth muscle, bovine aortic endothelial cells and rat aortic smooth muscle cells were cultured both separately and together in cocultures for 48 hr. Nitroprusside (1 mM) increased intracellular cGMP concentration 30-fold in smooth muscle cells (from a basal level of 103 +/- 54 fmol/mg of cell protein to 2920 +/- 1800 fmol/mg) but only 2-fold in endothelial cells (from 41 +/- 7 fmol/mg to 93 +/- 23 fmol/mg). When endothelial and smooth muscle cells were cocultured as a mixed cell population (1:1 cell ratio), both basal and nitroprusside-stimulated cGMP levels were significantly increased (550 +/- 250 and 13,240 +/- 9950 fmol/mg of total cell protein, respectively). The calcium ionophore A23187 (10 microM) caused no increase in cGMP concentration in either cell type cultured alone but produced a 6-fold increase in cocultures. Neither aspirin nor 5,8,11,14-icosatetraynoic acid influenced these results. No changes in cAMP levels were detected. Using cocultures in which one cell type was grown on microcarrier beads, we have shown that cGMP increased only in vascular smooth muscle cells and was not dependent upon the formation of junctions between endothelium and smooth muscle cells. In long-term (48-hr) mixed-cell cocultures, but not in short-term microcarrier cocultures, amplification of the nitroprusside-induced increase in cGMP was observed. These results show that responses associated with endothelium-dependent relaxation can be reconstituted in cultured endothelial and vascular smooth muscle cells and that endothelium generates a humoral factor(s) that stimulates accumulation of smooth muscle cGMP and has a longer-term effect that amplifies guanylate cyclase stimulation by nitroprusside, a drug acting directly upon smooth muscle to stimulate formation of the cyclic nucleotide. Cultured cells provide a valuable model system for the study of endothelium-vascular smooth muscle interactions.