In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI₂. Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI₂, hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI₂-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pH_i (11 ± 6 vs. 32 ± 10% in the absence and presence of 10¹⁰ M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 ± 9 vs. 41 ± 5% in the presence of 10¹² and 10⁹ M iloprost, respectively). Rapid decreases in pH_i stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pH_i. We conclude that PGI₂ receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI₂ does not involve inhibition of a pertussis toxin-sensitive G protein.