1. Vascular effects of substance P were studied in human isolated pial arteries removed from 14 patients undergoing cerebral cortical resection. 2. Substance P induced a concentration-dependent relaxation in the presence of indomethacin. No relaxation was seen in arteries where the endothelium had been removed. 3. N omega-nitro-L-arginine (L-NOARG, 0.3 mM) abolished the relaxation in arteries from six patients. The relaxation was only partially inhibited in the remaining eight patients, the reduction of the maximum relaxation being less than 50% in each patient. 4. The L-NOARG-resistant relaxation was abolished when the external K+ concentration was raised above 30 mM. 5. Substance P caused a smooth muscle hyperpolarization (in the presence of L-NOARG and indomethacin), but only when the artery showed an L-NOARG-resistant relaxation. 6. The results indicate that nitric oxide is an important mediator of endothelium-dependent relaxation in human cerebral arteries. Furthermore, another endothelium-dependent pathway, causing hyperpolarization and vasodilatation, was identified in arteries from more than half the population of patients.