Comparison of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat cultured vascular smooth muscle cells (VSMC) with those in rat gel-filtered platelets (GFP) was carried out using a receptor-ligand binding assay. The binding of each of three radiolabeled TXA2/PGH2 receptor antagonists [( 3H]S-145,[3H]SQ29,548, and [3H]ONO3708) displayed high affinity and specificity as well as saturable and displaceable binding to a single class of recognition sites with the same maximum number in both VSMC and GFP. The Kd values for [3H]S-145 were almost identical for both VSMC and GFP, whereas the values for [3H]SQ29,548 and [3H]ONO3708 for VSMC were approximately two and six times larger than that for GFP. Kinetic analysis of the binding of each receptor antagonist revealed a smaller K1 value (the association rate constant) for [3H]SQ29,548 and a larger K-1 value (the dissociation rate constant) for [3H]ONO3708 for VSMC compared to GFP, in contrast with almost the same kinetic constants for the [3H]S-145 binding for both cells. Comparison of the inhibitory potencies (Ki values) for [3H]S-145 binding for both VSMC and GFP proved that S-145 had the same affinity for both cells; ONO11120 and BM13177, as well as SQ29,548 and ONO3708, possessed lower affinity for VSMC; and U46619 exhibited higher affinity for VSMC. The rank orders of potency were identical in both cells (S-145 greater than SQ29,548 greater than ONO3708 greater than ONO11120 greater than BM13177), which correlated well with their pharmacological activities. These results suggest a similarity in ligand binding specificity with some differences in the accessibility of the antagonists in the TXA2/PGH2 receptors between platelets and vascular smooth muscles.