We investigated whether prostaglandins regulate endothelial nitric oxide synthase (eNOS) in the pig cerebral vasculature during the neonatal period. Prostaglandins, eNOS mRNA, eNOS protein, and NO production were higher in cerebral microvessels of newborn (1 day old) than in those of adult (6- to 8-month-old) pigs. The treatment of isolated cerebral microvessels of newborn animals with ibuprofen for 24 h reduced eNOS mRNA and nitrite production to levels in the adult; this effect of ibuprofen was prevented by concurrent treatment with prostaglandin (PG)E₂ analog 16,16-dimethyl-PGE₂, nonselective PGE₂ receptor analog 11-deoxy PGE₁, and prostaglandin EP₃receptor agonists sulprostone and M&B 28,767 but was not modified by PGI₂ analog carbaprostacyclin, PGD₂, and EP₁ receptor agonist 17-phenyl trinor PGE₂. Correspondingly, 16,16-dimethyl-PGE₂ and M&B 28,767 increased eNOS mRNA expression of adult microvessels to values in the newborn. Data similar to those with isolated cerebral vessels were obtained through histochemical analysis (NADPH-diaphorase positivity) of brain from newborn animals treated in vivo with ibuprofen in combination or not with sulprostone. Furthermore, substance P-induced NO-mediated cerebral vasorelaxation was decreased to adult values through the treatment of newborn pigs with ibuprofen; this effect was prevented by concomitant treatment with sulprostone. It is concluded that PGE₂ regulates eNOS in newborn pig cerebral microvessels via EP₃ receptors; this may be physiologically required during normal neurovascular development.