Both acetylcholine (ACh) and 5-hydroxytryptamine (5HT) are used to examine nitric oxide (NO)-mediated vasodilatation in humans. Animal data suggest that both substances can also induce the release of prostacyclin (PGI ). This study was designed to investigate the role of the prostaglandin pathway in Ach- and 5HT-induced vasodilation in humans. The experiments were done in three groups of healthy male volunteers. In group 1 (n = 6), ACh (100-1,000 ng/kg/min) and sodium-nitroprusside (10-100 ng/kg/min) were infused into the brachial artery alone, together with a continuous infusion of indomethacin (1.3 micro g/kg/min) and during a combined infusion of indomethacin and the competitive NO synthase inhibitor N -monomethyl-l-arginine (l-NMMA; 30 micro g/kg/min). In group 2 (n = 5), 5HT (0.3-1.0 ng/kg/min) was infused alone and together with a continuous infusion of indomethacin and l-NMMA. In group 3 (n = 6), the synthetic prostaglandin analog iloprost (0.5-4.5 ng/kg/min) was infused together with a continuous infusion of saline, l-NMMA, and l-NMMA with indomethacin, respectively. The infusions of indomethacin and l-NMMA started 10 min before the infusion of ACh, 5HT, iloprost, and sodium nitroprusside. Forearm blood flow was measured using computerized venous occlusion plethysmography. Both the Ach- and 5HT-induced vasodilator responses were significantly attenuated by indomethacin (p < 0.05 for both), but not further influenced by a concomitant infusion of l-NMMA. The vasodilatation induced by iloprost was significantly inhibited by l-NMMA (p < 0.05) and not affected by indomethacin. The sodium nitroprusside-induced vasodilation was influenced by neither l-NMMA nor indomethacin. It is concluded that in the human forearm, the prostaglandin pathway is involved in both the Ach- and 5HT-induced NO-mediated vasodilatation.