Prostaglandin (PG) D(2) 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 [(3)H]PGD(2) as ligand. [(3)H]PGD(2) binding to intact platelets was saturable and rapid with the ligand bound by 3 min at 20 degrees C. PG competed with the [(3)H]PGD(2) binding site with a potency series: PGD(2) (IC(50) = 0.08 muM) >> PGI(2) (IC(50) = 2 muM) > PGE(1) (IC(50) = 6 muM) > PGF(2alpha) (IC(50) = 8 muM). 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 PGD(2) 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 PGD(2) on aggregation and adenylate cyclase activity (1978. Blood.52: 618-626.). In the presence of 50 nM [(3)H]PGD(2), the patients' platelets bound 7.1+/-2.9 fmol ligand/10(8) platelets compared with 15.1+/-1 fmol/10(8) platelets in normals, a decrease of 53% (P < 0.01). Scatchard analysis showed that the K(d) of [(3)H]PGD(2) binding (33 nM) was comparable to normal platelets, which indicates that the decreased PGD(2) binding in these platelets represented fewer receptors rather than altered affinity of the ligand for the binding site. The 53% decrease in [(3)H]PGD(2) binding correlated with a 48% decrease in PGD(2)-activated platelet adenylate cyclase. The characterization of the platelet PGD(2) binding site provides further direct evidence that there are at least two PG receptors on platelets, one for PGE(1) and PGI(2), and a separate receptor for PGD(2). 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 PGD(2) binding sites in patients with myeloproliferative disorders.