PCO₂ in the lumen and serosa of cecum and colon was measured in rats, guinea pigs, and dogs to examine the relationship between serosal PCO₂ and the incidence of intestinal necrotic lesions after administration of gas-carrier contrast agents in rodents. The effects of the dietary substrate were tested in a group of mice maintained on a diet based on glucose as the only carbohydrate source. The anesthetic used was a fentanyl-fluanison-midazolam mixture (rodents) and pentobarbital (dogs). PCO₂ was measured in vivo and postmortem, and the kinetics of the postmortem serosal PCO₂ [transmural CO₂ flux (J_CO2)] was calculated. PCO₂ in the cecal serosa and lumen, respectively, was 64 ± 4 and 392 ± 18 Torr in rats, 67 ± 3 and 276 ± 17 Torr in guinea pigs, and 73 ± 6 and 137 ± 7 Torr in mice on glucose-based diet. In the colon serosa and lumen of dogs, PCO₂ was 30 ± 6 and 523 ± 67 Torr, respectively. Serosal PCO₂ increased rapidly after death in rats and slower in guinea pigs and mice, and the slowest change was observed in dogs. Compared with dogs, serosal PCO₂ and J_CO2 of rats and guinea pigs were significantly higher. Serosal PCO₂ of guinea pigs was similar to that of rats, whereas the J_CO2 of guinea pigs was significantly lower. These data suggest a causal relationship between the ability of the cecal and colonic wall to act as a barrier to CO₂ diffusion and the presence of characteristic gas-carrier contrast agent-induced intestinal lesions in mice and rats and their absence in guinea pigs, dogs, and other species.