Platelets possess membrane receptors which mediate the aggregatory response to thromboxane A2 (TXA2) and prostaglandin H2 (PGH2). It has been observed recently that the affinities for a series of TXA2/PGH2 mimetics are decreased in crude human platelet membranes and solubilized membranes compared to intact washed platelets. The present study investigated the notion that platelets contain a substance that is released during platelet lysis that reduces the affinity of the TXA2/ PGH2 receptor for agonists. The displacement of 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4- hydroxyphenyl)-13,14-dihydro-13-aza-15αβ-ω-tetranor-TXA2 ([125I]PTA-OH), a TXA2/PGH2 receptor antagonist, from its binding site in intact washed platelets by TXA2/PGH2 mimetics and antagonists was characterized in the presence or absence of the supernatant (50,000g) obtained from sonicated platelets. In the presence of the supernatant, there was a significant (P < 0.025) increase in the ic50 values for the TXA2/PGH2 mimetics U46619, SQ26655, and ONO11113. The increase in the ic50 for U46619 induced by the supernatant was abolished by either boiling or treating the supernatant with trypsin. The supernatant did not affect the Kd or Bmax of [125I]PTA-OH or the ic50 of the TXA2/PGH2 antagonist, SQ29548. Pretreatment of the platelets with the supernatant resulted in a significant (P < 0.02) reduction in the aggregation response induced by U46619. Gel filtration (Sephacryl S200) of the supernatant revealed a fraction (molecular weight ~100,000 daltons) which significantly increased the ic50 for U46619 to displace [125I]PTA-OH from its binding site. Thus, human platelets appear to possess a protein(s) that is released into the supernatant upon sonication and inhibits the binding of TXA2/PGH2 agonists but not antagonists to their receptor. This protein may play a role in the regulation of platelet responses to the aggregatory stimuli TXA2/PGH2.