The effect of indomethacin on dilator prostanoid receptor-mediated cAMP formation was investigated using primary cultures of vascular smooth muscle cells from the newborn pig cerebral microvessels. Cerebral microvascular smooth muscle cells responded to dilator prostanoids (iloprost > PGE2) by increasing cAMP formation and release into the media (EC50 = 2 x 10(-8) M and 2 x 10(-7) M for iloprost and PGE2, respectively). Indomethacin inhibited iloprost- and PGE2-evoked increases in cAMP formation (IC50 = 10(-4) M) and release (IC50 = 10(-6) M) by microvascular smooth muscle cells (maximal inhibition 80-90%), whereas isoproterenol-induced cAMP formation was only slightly attenuated at the highest concentration of indomethacin used (10(-3) M). Aspirin was much less effective in inhibiting dilator prostanoid-induced cAMP formation and release by the cells. Direct analysis of prostacyclin receptor sites using [3H]iloprost as the ligand revealed saturable, high affinity (ED50 = 2 x 10(-8) M) and reversible binding to the membranes isolated from cerebrovascular smooth muscle cells. Indomethacin dose-dependently inhibited [3H]iloprost receptor binding (ID50 = 10(-4) M; maximal inhibition, 70%). The present data suggest that combination of highly effective inhibition of prostaglandin H synthase and receptor binding resulting in inhibition of dilator prostanoid-mediated cAMP formation in target cells may contribute to the increased efficacy of indomethacin compared with other prostaglandin H synthase inhibitors in blocking certain vasodilator responses associated with prostanoids.