The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated in part by changes in local production of 20-hydroxyeicosatetraenoic acid (20-HETE). In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle, and the role of 20-HETE in this responsiveness. Rats were fed either a normal salt (NS, 0.45%) or high salt (HS, 4%) diet for 4-5 weeks. There was no difference in steady-state tissue PO2 between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue PO₂ to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA ) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the KCa channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats, and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups, but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K_Ca channels, and that a high salt diet impairs arteriolar responses to increased oxygen availability due to a reduction vascular smooth muscle responsiveness to 20-HETE.