Pial arterioles on the surface of the mouse brain were observed in vivo under a chamber with a glass window. When placed under the window, calcium ionophore, acetylcholine, and previously acidified sodium nitrite each dilated the arterioles. If the cyclooxygenase inhibitors indomethacin or acetylsalicylic acid were first placed in the chamber, subsequent dilation of the arterioles by calcium ionophore was reduced to essentially zero. Similar blockade of cyclooxygenase failed to significantly reduce dilation by acetylcholine or sodium nitrite. We have previously shown that dilations by calcium ionophore and acetylcholine were endothelium dependent. Our present experiments show that the endothelium-dependent mechanism for dilation by calcium ionophore is cyclooxygenase dependent, while that for acetylcholine is not. This implies that, in pial arterioles, the endothelium-derived relaxing factor for acetylcholine differs from that for calcium ionophore. This agrees with data from other microvascular beds. I n mouse and cat cerebral microvessels on the surface of the brain (pial arterioles), we have demonstrated endothelium-dependent relaxation and constriction in vivo.1 -3 Instead of directly relaxing vascular smooth muscle, some (but not all) dilators relax these arterioles by causing their endothelium to release a relaxing mediator. The dilators for which we have shown this phenomenon are calcium ionophore A-23187, acetylcholine (ACh), and bradykinin. These same dilators have been shown by others to produce endothelium-dependent relaxation of large conductance vessels in vitro. 4 -5 In conductance arteries, there is evidence that the endothelium-derived relaxing factor (EDRF) is the same for all three dilators. 6 Moreover, this EDRF (sometimes called classical EDRF) is not cyclooxygenase dependent and hence relaxation by the three dilators is not blocked by cyclooxygenase inhibitors such as indomethacin.1 -4 However, in large vessels, prostacyclin (a product of cyclooxygenase activity) is also recognized as an EDRF, albeit not the classical EDRF. Moreover, there may be other EDRFs released from other large vessels.