Prostaglandin (PG) D₂ is synthesized in platelets at concentrations which could inhibit aggregation via activation of adenylate cyclase. To more directly define platelet-PG interactions, a binding assay has been developed for platelet PG receptors with [³H]PGD₂ as ligand. [³H]PGD₂ binding to intact platelets was saturable and rapid with the ligand bound by 3 min at 20°C. PG competed with the [³H]PGD₂ binding site with a potency series: PGD₂ (IC₅₀ = 0.08 μM) ≫ PGI₂ (IC₅₀ = 2 μM) > PGE₁ (IC₅₀ = 6 μM) > PGF_2α (IC₅₀ = 8 μM). Scatchard analysis of binding data from six normal subjects showed a single class of binding sites with a dissociation constant (K_d) of 53 nM and 210 binding sites per platelet. This PGD₂ receptor assay was then used to study platelets from five patients with myeloproliferative disorders (polycythemia vera, essential thrombocythemia, and chronic myelogenous leukemia), as over 90% of these patients have platelets resistant to the effects of PGD₂ on aggregation and adenylate cyclase activity (1978. Blood.52: 618-626.). In the presence of 50 nM [³H]PGD₂, the patients' platelets bound 7.1±2.9 fmol ligand/10⁸ platelets compared with 15.1±1 fmol/10⁸ platelets in normals, a decrease of 53% (P < 0.01). Scatchard analysis showed that the K_d of [³H]PGD₂ binding (33 nM) was comparable to normal platelets, which indicates that the decreased PGD₂ binding in these platelets represented fewer receptors rather than altered affinity of the ligand for the binding site. The 53% decrease in [³H]PGD₂ binding correlated with a 48% decrease in PGD₂-activated platelet adenylate cyclase. The characterization of the platelet PGD₂ binding site provides further direct evidence that there are at least two PG receptors on platelets, one for PGE₁ and PGI₂, and a separate receptor for PGD₂. Direct binding analysis will be a useful tool for studying the role of PG in regulating platelet function, as demonstrated by the selective loss of PGD₂ binding sites in patients with myeloproliferative disorders.