1. The effect of severe hypoxia on force, intracellular Ca2+ concentration ([Ca2+]i) and pHi was studied in isolated small arteries from rat brain and rat mesenterium. The arteries were mounted for isometric force recording while [Ca2+]i was measured with fura-2 or pHi was measured with bis-carboxyethylcarboxyfluorescein (BCECF). 2. Hypoxia reduced the force development in response to arginine vasopressin (AVP) while [Ca2+]i was unchanged or only slightly reduced. Inhibition of acid extrusion by omission of sodium caused no force development in mesenteric arteries, but the fall in pHi was enhanced during hypoxia. In cerebral arteries, hypoxia reduced the force development associated with omission of sodium, and the fall in pHi was less than during normoxic conditions. When acid extrusion was intact, pHi was not affected by hypoxia and the changes in pHi during activation with AVP were similar during hypoxia and in the control situation. 3. Although a decrease in smooth muscle [Ca2+]i may be partly responsible for the reduced force development during hypoxia, [Ca2+]i-independent mechanism(s) may play an even more important role. Furthermore, although hypoxia and force development are associated with enhanced acid production, acid extrusion maintains pHi near the control level and it is unlikely that a decrease in smooth muscle pHi plays any role in the reduced force development during hypoxia.