The binding of glycosaminoglycans to intact washed human platelets was studied. The platelet binding of a 3H-labeled unfractionated heparin was saturable and reached equilibrium in 10-15 minutes. Heparin binding was specific: a 50-fold molar excess of an equivalent unlabeled heparin displaced up to 90% of labeled heparin, while chondroitin sulfate A and hyaluronic acid minimally displaced the binding of labeled heparin. Low molecular weight heparin fragments showed intermediate efficacy in displacing the binding of unfractionated [3H]heparin. Dextran sulfate (Mr 8,000, sulfation 17%) was as potent as unfractionated heparin in displacing binding, while neutral dextrans were ineffective. We observed that platelet activation by the calcium ionophore A23187 increased heparin binding by 2 to 3-fold, principally by enhancement of binding capacity not binding affinity. This process of heparin binding to the platelet surface may mediate some of the reported effects of heparin on platelet behavior.