The present study of newborn pig cerebral circulation investigated the role of pertussis toxin (PTX)-sensitive GTP binding proteins in the permissive action of prostacyclin in specific dilator responses. Pial arterioles of anesthetized piglets were observed through closed cranial windows. The piglets were treated topically with PTX and intravenously with indomethacin. The effects of hypercapnia (10% CO₂ ventilation) and topical 5,6-epoxyeicosatrienoic acid (5,6-EET) on pial arteriolar diameter were noted before and after the intervention. Samples of the artificial cerebrospinal fluid (aCSF) were collected from beneath the cranial windows for determination of the cAMP concentration. After administration of PTX, indomethacin still abolished pial arteriolar dilation to both hypercapnia and 5,6-EET and also inhibited the cAMP elevation caused by hypercapnia. The addition of phorbol 12-myristate 13-acetate (PMA), but not iloprost, restored the increase in cAMP and vascular responses to hypercapnia and 5,6-EET. Therefore, in the newborn pig cerebral microvasculature, PTX appears to inhibit a G protein involved in the permissive action of prostacyclin. However, the protein kinase C (PKC) activator PMA appears to act downstream from the block, and, therefore, the permissive action of PMA is not affected by PTX. We suggest that the prostacyclin IP receptor may be coupled to phospholipase C via a PTX-sensitive G protein that normally permits vasodilation to specific stimuli via activation of a PKC, resulting in phosphorylation of a component of the adenylyl cyclase pathway.