The proposal that potassium is an endothelial derived hyperpolarizing factor (EDHF) has focussed attention on the effects of elevated extracellular potassium ([K⁺]_o) on arterial tone. Results from different laboratories show that rat isolated mesenteric arteries do not reliably relax to an increase in [K⁺]_o with no consensus for that discrepancy. The aim of this study was to define the experimental factors that determine the response of rat mesenteric artery to elevated [K⁺]_o. Male Wistar rats (200-250 g) were killed humanely and the superior mesenteric arterial tree removed. For isometric tension recording, sections of third order arteries were mounted in a Mulvany-type myograph containing bicarbonate buffered physiological salt solution (PSS) at 37 ^oC. We have previously shown that relaxation of phenylephrine (PE) induced force by raised [K⁺]_o was dependent upon extracellular sodium, being attenuated in reduced sodium PSS and enhanced, sodium-dependently, by monensin (Brochet et al., 2002). Relaxation was independent of the endothelium and sensitive to ouabain, with 100 μM being required for full inhibition (Brochet and Langton, 2003). In this study, we found that the concentration of PE had no effect on the relaxation of arteries to an increase in [K⁺]_o. In addition, inhibition of BKCa by TEA (5 mM), charybdotoxin (100 nM) or iberiotoxin (100 nM) did not induce a relaxation when [K⁺]_o was increased from 5.9 to 13.8 mM. The hyperpolarization and relaxation to an increase in [K⁺]_o from 4.6 to 13.8 mM was enhanced by increasing the duration of exposure to PE. Moreover, the time dependence of the effect of PE was attenuated in the presence of diphenylboric acid 2-aminoethyl ester (2-APB; 75 μM), an IP₃ receptor inhibitor, and abolished by SKF96365 (10 μM), a store-operated cation channel (SOC) inhibitor. In contrast, 50 nM ryanodine, a concentration previously shown to induce the Ca²⁺ release from intracellular stores (Meissner, 1986), enhanced the relaxation to raised [K⁺]_o. These data suggest that accumulation of [Na⁺]_i following activation of SOCs by PE and the depression of Na,K-ATPase activity in low [K⁺]_o both serve to potentiate the upturn in the activity of the Na,K-ATPase when [K⁺]_o is subsequently increased. This results in hyperpolarization and relaxation of the arterial smooth muscle.