In the present study we investigated the mechanism of prolongation of the plasma retention of free hemoglobin by polymerization. Polymerization of intramolecularly crosslinked hemoglobin with glutaraldehyde yields a mixture of large polymers, small polymers and monomers. In exchange transfusion experiments in rats we analyzed plasma samples by gel filtration to determine the clearance of polymers of different size. A positive correlation was found between polymer size and vascular retention. Furthermore, the clearance of large polymers appeared to be highly dose-dependent: after 20% and 70% exchange transfusions, we observed for large polymers a plasma half-life of 12 and 26 hours, respectively, whereas the half-life for 64kD monomers was 4 hours in both cases. The degradation of hemoglobin was followed by measuring the bilirubin excretion. The infused heme was recovered as bilirubin within 72 hours. The delay between the disappearance of free hemoglobin from the plasma and the recovery as bilirubin was about six hours and was not affected by polymerization or dose. We conclude that polymerization prevents the operation of certain clearance mechanisms, while still allowing a route of clearance that is easily saturated. The intracellular degradation of heme into bilirubin is not affected by the modifications of hemoglobin and is not easily saturated.