One of the major contributory factors to hyperbilirubinemia in the human infant is bilirubin production. Determination of the pulmonary excretion rate of carbon monoxide is used to measure indirectly the rate of bilirubin production by estimating the endogenous production of CO, which is formed in equimolar amounts with bilirubin in the catabolism of heme. There has been no study to confirm that complete recovery of CO produced in vivo from the catabolism of known amounts of heme occurs via pulmonary excretion. We report here quantitative determinations of the pulmonary excretion rate of CO in five adult male Wistar rats after injection of known amounts of heme in the form of red blood cells "damaged" by incubation in a solution containing a sulfhydryl inhibitor, NEM. The mean recovery of "extra" CO above baseline production represented as a molar ratio of extra CO to heme was 0.98 +/- 0.02 (S.E.). Control studies showed no extra CO production after starvation, injection of NEM in an amount comparable to that used in the experimental animals, or injection of undamaged red blood cells.