We investigated the etiology of thrombocytopenia, with or without platelet thrombi, occurring while patients are receiving parenteral heparin. We used two in vitro methods to detect possible humoral factors in the sera of patients who became thrombocytopenic while receiving heparin. In the presence of heparin, four of four such patients' serum caused platelet aggregation. Only serum from the patient most severely affected clinically caused release of platelet factor 3 (PF3). All control sera gave negative results by both methods. We propose that platelet aggregation studies may be a sensitive and reliable method of confirming that thrombocytopenia occurring during heparin therapy is due to a humoral factor requiring the presence of heparin.