Exposure of conscious animals to environmental heat stress increases portal venous radical content. The nature of the observed heat stress-inducible radical molecules suggests that hyperthermia produces cellular hypoxic stress in liver and intestine. To investigate this hypothesis, conscious rats bearing in-dwelling portal venous and femoral artery catheters were exposed to normothermic or hyperthermic conditions. Blood gas levels were monitored during heat stress and for 24 h following heat exposure. Hyperthermia significantly increased arterial O2 saturation, splanchnic arterial-venous O2 difference, and venous PCO2, while decreasing venous O2 saturation and venous pH. One hour after heat exposure, liver glycogen levels were decreased approximately 20%. Two hours after heat exposure, the splanchnic arterial-venous O2 difference remained elevated in heat-stressed animals despite normal Tc. A second group of rats was exposed to similar conditions while receiving intra-arterial injections of the hypoxic cell marker 3Hmisonidazole. Liver and intestine were biopsied, and 3Hmisonidazole content was quantified. Heat stress increased tissue 3Hmisonidazole retention 80% in the liver and 29% in the small intestine. Cellular 3Hmisonidazole levels were significantly elevated in intestinal epithelial cells and liver zone 2 and 3 hepatocytes and Kupffer cells. This effect was most prominent in the proximal small intestine and small liver lobi. These data provide evidence that hyperthermia produces cellular hypoxia and metabolic stress in splanchnic tissues and suggest that cellular metabolic stress may contribute to radical generation during heat stress.