Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can be cleaved by thrombin. As yet, however, little is known from studies of the receptor protein itself. To obtain such information, we have prepared monoclonal antibodies against a peptide corresponding to receptor residues Ser42 through Phe55, the domain immediately distal to the site of cleavage by thrombin. By flow cytometry, all of the antibodies reacted with the thrombin-responsive megakaryoblastic CHRF-288 and HEL cell lines, but not with the T-lymphoid Sup-T1 cell line. Functionally, the antibodies inhibited platelet responses to alpha-thrombin, gamma-thrombin, and trypsin, but had no effect on platelet activation by ADP, epinephrine, or the thromboxane analog U46619. Radioiodinated antibody bound to approximately 1,800 sites/platelet, a value similar to the reported number of moderate affinity thrombin binding sites per platelet. On Western blots, the antibodies recognized a 66-kDa protein in platelet, HEL, and CHRF-288 membranes. The discrepancy between this apparent size and the predicted mass of the receptor suggests that, as with other G protein-coupled receptors, one or more of the potential sites for N-linked glycosylation have been utilized. Therefore, these results suggest that: 1) the cloned thrombin receptor is involved in a broad range of platelet responses to thrombin, as well as gamma-thrombin and trypsin; 2) as predicted, the N terminus of the receptor is accessible on the platelet surface; 3) the moderate affinity thrombin binding site noted in earlier studies may be the receptor; 4) potentially as much as one third of the mass of the receptor is carbohydrate.