Cultured endothelial cells isolated from human umbilical vein bind heparin and heparin fragments. The binding capacity of endothelial cells for 35S-heparin was for 38% composed of high affinity binding sites (Kd = 11 X 10(-8) M) and for 62% of sites with much lower affinity (Kd = 14 X 10(-7) M). The affinity of unlabeled compounds for heparin binding sites was determined by competition with binding of 125I-heparin. I50 found for unlabeled heparin was 16 X 10(-8) M, which is in agreement with the Kd for binding of heparin to high affinity sites. PK 10169, a low molecular weight fragment of heparin, competed only at relatively high concentrations (I50 = 10(-5) M). Competition experiments with subfractions of PK 10169 showed that I50 was inversely correlated with molecular weight. Gelfiltration of 35S-heparin and 35S-PK 10169 before and after binding to endothelial cells demonstrated a selective binding of high molecular weight molecules from polydisperse heparin and PK 10169 preparations. Bound heparin and PK 10169 molecules were detached from the cell-surface by proteolytic treatment and tested for antifactor-Xa and antifactor-IIa activity. Released heparin is slightly more active in antifactor-Xa and antifactor-IIa activity than its parent preparation. Released PK 10169 was 4 fold more active in antifactor-Xa and 8 fold more active in antifactor-IIa assays than heparin.