Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 ± 0.05 (P < 0.001 different from unity) (90 ± 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with K(d) of 234 ± 103 pM, B(max) of 0.7 ± 0.3 pM/mg protein (180 ± 87 sites/WP), and a lower affinity site with K(d) of 2.31 ± 0.86 nM, B(max) of 2.2 ± 0.3 pM/mg protein (666 ± 65 sites/WP). [125I]BOP association and dissociation kinetics gave a K(d) of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 ± 24 pM, shape change was 263 ± 65 pM, and aggregation was 4.4 ± 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two K(d)s for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.