Endothelium-dependent constriction in mouse pial arterioles was demonstrated by testing contractile responses before and after endothelial injury. All responses were monitored at the same site as the injury. Injury was produced in vivo by exposing an arteriole on the brain surface to the beam of a 6-mW helium-neon laser after first sensitizing the microvascular bed to the laser energy by injecting Evans blue intravenously. The contractile response to serotonin creatinine sulfate (20 micrograms/ml) and to sodium arachidonate (30 micrograms/ml) was monitored in vivo with an image splitter and TV microscope. The responses before laser injury were always constriction; the responses after laser injury were always relaxation. After laser injury, acetylcholine chloride (80 micrograms/ml) constricted every vessel tested at the injured site. Thus, the injured segment had not lost the capacity to constrict even though neither serotonin nor arachidonate remained able to induce a constriction. The endothelium-dependent constrictions to serotonin or arachidonate were also blocked by pretreatment of the mouse with cyclooxygenase inhibitors, acetylsalicylic acid (100 mg/kg i.p.) or indomethacin (5 mg/kg). The data suggests that endothelium-dependent constriction to serotonin is mediated by release of arachidonate from the endothelial cell and conversion of that arachidonate by cyclooxygenase to some constricting prostanoid.