Heparin-induced thrombocytopenia (HIT) is caused by antibodies (HIT-Abs) that bind to a complex of heparin and platelet factor 4. We investigated the epitope specificity of the HIT-Abs, and found that the HIT-Abs recognized solid-phase immobilized complexes with an optimum ratio of four to eight molecules of PF4 per molecule of heparin. To try to define the epitopes within the PF4 molecule, intact and reduced (linearized) PF4 was tested against 29 different sera from patients with HIT. In addition, eight different peptides that spanned the PF4 molecule were studied for their ability to bind to the HIT-Abs either alone or in the presence of heparin. With the exception of a subpopulation of patient samples (5/29, 17%), we found that reduced PF4 and the peptides were uniformly non-reactive with the HIT-Abs in the presence of heparin. Reduced PF4 and PF4 carboxy-terminal peptides with a minimum size of 19 amino acids were recognized by a minority (5/29) of HIT-Abs samples but only when heparin was present. The specificity of this subgroup of samples from patients with HIT was highly restricted and the loss of one amino acid (peptide reduced in length from 19 to 18 amino acids) rendered the peptides non-reactive. The clinical characteristics of these patients were similar to the other HIT patients. These studies demonstrate that the majority of HIT-Abs recognize a noncontiguous conformational epitope on the PF4 molecule that is produced when four to eight PF4 molecules are bound together by heparin.