Endothelium-derived cyclooxygenase (COX) products regulate cerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebral microvessels. We investigated the role of protein phosphorylation in the regulation of COX activity. The protein tyrosine phosphatase (PTP) inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acid rapidly stimulated COX activity, whereas the protein tyrosine kinase inhibitors, genistein and tyrphostins, inhibited it. Protein synthesis inhibitors did not reverse the stimulation of COX activity evoked by PTP inhibitors. Similar changes were observed in other vascular cells from newborn pigs that also express COX-1 and COX-2 (cerebral microvascular smooth muscle cells and aortic endothelial cells) but not in human umbilical vein endothelial cells or Swiss 3T3 fibroblasts that express COX-1 only. Tyrosine-phosphorylated proteins were immunodetected in endothelial cell lysates. COX-2 immunoprecipitated from ³²P-loaded endothelial cells incorporated ³²P that was increased by PTP inhibitors. COX-2, but not COX-1, was detected in endothelial fractions immunoprecipitated with anti-phosphotyrosine. These data indicate that tyrosine phosphorylation posttranslationally regulates COX activity in newborn pig vascular cells and that COX-2 is a substrate for phosphorylation.