Antibodies to PF4/heparin can be demonstrated in almost all patients with heparin-induced thrombocytopenia/thrombosis (HIT/HITT) and in some persons exposed to heparin who do not have clinical manifestations. The role of anti-PF4/heparin antibodies in the pathogenesis of HIT/HITT has been difficult to establish because the antibodies found in serum are generally polyclonal and polyspecific. To circumvent this problem, we developed a murine monoclonal antibody (mAb) to human (h) PF4/heparin complexes. A monoclonal IgG_2bκ antibody (designated KKO) was identified that bound specifically to hPF4/heparin complexes. Maximal binding of KKO to hPF4/heparin complexes occurred at similar molar ratios of PF4:heparin observed for HIT/HITT antibodies. KKO also bound to hPF4 in association with other glycosaminoglycans. Platelet activation by KKO required heparin and was abrogated by blockade of FcγRIIA. In the presence of PF4, KKO bound to endothelial cells, but not to CHO cells lacking heparan sulfate proteoglycans. Variants of PF4 complexed to heparin were recognized equally well by KKO and HIT/HITT sera. KKO competes for binding with a subset of HIT/HITT antibodies that are relatively spared by mutations in the 3rd domain of PF4. The nucleotide and predicted amino acid sequences of KKO and RTO, a murine anti-hPF4 mAb that does not require heparin for binding, revealed no obvious relationship in either the heavy- or the light-chain immunoglobulin variable regions. These studies suggest that KKO recapitulates the antigenic and functional specificity of a subset of HIT/HITT antibodies and may, therefore, provide insight into the pathogenesis of thrombocytopenia and thrombosis in affected persons.