Thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors were characterized in rat vascular smooth muscle cells (VSMC). The specific binding of [3H]SQ 29,548 was inhibited by KW-3635, a novel non-prostanoic TXA2 antagonist, SQ 29,548 and BM-13505 (daltroban). SQ 29,548 showed a single class of binding sites with a Ki value of 1.6 nM. The inhibition patterns were better fit to two-component curves for KW-3635 (Ki values of 0.45 nM and 42 nM) and BM-13505 (2.3 nM and 20 nM). U46619, a TXA2 agonist, induced an increase in intracellular calcium concentration ([Ca2+]i), which was inhibited by these antagonists. KW-3635 and SQ 29,548 did not induce any increase in [Ca2+]i, whereas BM-13505 was found to induce a smaller increase in [Ca2+]i. The BM-13505-induced increase in [Ca2+]i was also inhibited by pretreatment with KW-3635, SQ 29,548 and BM-13505. The results demonstrate that BM-13505 has partial agonistic activity on TXA2/PGH2 receptors, and KW-3635 and SQ 29,548 do not. SQ 29,548 and BM-13505 inhibited both U-46619- and BM-13505-induced increases in [Ca2+]i to a similar degree. Alternatively, KW-3635 inhibited a U46619-induced increase in [Ca2+]i more effectively than a BM-13505-induced increase. These results suggest the heterogeneity of functional binding sites or subtypes of TXA2/PGH2 receptors present in VSMC.