1. Hypoxia (P(O2) < 5 mmHg) decreased vessel tone in isolated rat mesenteric arteries precontracted with either high [K+] or the thromboxane analogue U46619. This response was not altered by N-nitro-L-arginine (L-NA) and indomethacin. Simultaneous measurement of pH(i) and tension showed that the decrease in vessel tone was accompanied by an intracellular acidification. Similar reductions in tone and pH(i) were observed with the metabolic inhibitors 2,4-dinitrophenol (DNP) and sodium azide. 3. The presence of the lactate transport inhibitor α-cyano-4-hydroxy-cinnamic acid (CHC) increased the magnitude of the acidification and resulted in a significantly faster reduction in tone in response to hypoxia. Addition of CHC to normoxic tissues caused both a vasodilatation and a reduction of pH(i). 4. A decrease in pH(i) induced on washout of ammonium chloride (NH4Cl) resulted in an increase in tone. 5. Relaxation to hypoxia or metabolic inhibition was unaffected when the change in pH(i) was neutralized by addition of the weak base trimethylamine (TMA). 6. It is concluded that severe hypoxia decreases tone in isolated rat mesenteric arteries by a mechanism which is independent of nitric oxide and prostaglandins. Both severe hypoxia and metabolic inhibition reduced pH(i), although this does not appear to be contributing to the changes in tone observed.