Extracellular-superoxide dismutase (EC-SOD) is heterogenous in the vasculature with regard to heparin affinity and can be separated into three fractions: A, without affinity; B, with weak affinity; and C, with relatively strong heparin affinity. The plasma clearance of intravenously injected 125I-labeled and unlabeled human EC-SOD C was studied in rabbits. About 90% of injected 125I-EC-SOD C was eliminated from the blood within 5-10 min. Injection of heparin after 10 or 20 min led to an immediate release of all sequestered 125I-EC-SOD C back to the blood plasma. Later injections of heparin led to diminished release, although release could still be demonstrated after 72 h. A half-time of approximately 10 h could be calculated for heparin-releasable 125I-EC-SOD C. Unlabeled EC-SOD C, determined as enzymic activity and with ELISA, was likewise sequestered and released to the same degree as 125I-labeled EC-SOD C by heparin as tested at 20 min and 5 h. The immediacy of the heparin-induced release indicates that the sequestered enzyme had been bound to endothelial cell surfaces. The length of the half-time suggests that the putative cell surface binding has a physiological function and is not primarily a step in enzyme degradation. The distribution of sequestered 125I-labeled EC-SOD C to different organs was determined at times between 10 min and 24 h. Of the organs, the liver contained the most 125I-EC-SOD C, followed by kidney, spleen, heart, and lung. At all investigated times, the content in the analyzed organs was nearly as large as the amount that could be promptly released to plasma by intravenous heparin. This indicates that almost all 125I-EC-SOD C in the organs was present on endothelial cell surfaces and was not bound by other tissue cell surfaces, or was present within the cells.