Acetylcholine-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K⁺, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin, were contracted with phenylephrine. Acetylcholine (10^-10-10^-6M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition, the KCa channel inhibitors apamin (100 nM) or charybdotoxin (100 nM), and eliminated by the combination of apamin plus charybdotoxin. Relaxations to acetylcholine were also blocked by a combination of barium (200 µM) and apamin, but not barium plus charybdotoxin. Addition of K⁺ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K⁺ addition prior to acetylcholine restored charybdotoxin-sensitive component of relaxations to acetylcholine. K⁺ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium, but not by charybdotoxin and apamin. Using whole-cell patch-clamp analysis, acetylcholine (10^-7 M) stimulated voltage-dependent outward K⁺ current from endothelial cells, which was inhibited by charybdotoxin indicating K⁺ efflux. Arachidonic acid (10^-7-10^-4M) induced concentration-related relaxations that were inhibited by apamin, but not by charybdotoxin and barium. Addition of arachidonic acid after K⁺(10.9 mM) resulted in more potent relaxations to arachidonic acid compared to control without K⁺(5.9 mM). These findings suggest that in rabbit mesenteric arteries, acetylcholine-induced, L-NA and indomethacin-resistant relaxation is mediated by endothelial cell K⁺ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.