We studied the effect of tin protoporphyrin, a potent inhibitor of heme oxygenase (EC 184.108.40.206), on carbon monoxide (CO) production in mature mice. Measurements of CO production provide a sensitive, noninvasive means of quantitating heme catabolism. CO accumulation in the gas space of closed chambers was decreased by about 25% for mice treated with two 50 nmol/g doses of tin protoporphyrin as compared to saline-treated controls. Calculated rates of CO production were 0.28 ± 0.07 and 0.40 ± 0.05 nmol-g-1h-1 for mice injected with tin protoporphyrin and saline, respectively (p < 0.01). When hemin (125 nmol/g) was administered to simulate hemolysis, CO production increased markedly in both saline- and tin protoporphyrin-treated mice. However, the rate of CO production in tin protoporphyrin-treated mice was only 44% that of saline-treated animals (p < 0.0001). These studies demonstrate that tin protoporphyrin inhibits heme catabolism in both the basal- and heme-loaded states and confirm that this inhibition is at the heme oxygenase step in the heme to bilirubin pathway.