The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1alpha,2alpha (Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [3H]SQ 29,548 [[1S-[1alpha,2alpha (Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TPalpha- and TPbeta-transfected COS-7 cells. These isoforms possess a different tail length, the alpha being shorter than the beta isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC50 value for PBT-3 = 2.0 +/- 0.3 x 10-7 M was observed for TPalpha, whereas this was one-sixth less active on the TPbeta isoform (IC50 = 1.2 +/- 0.2 x 10-6 M), suggesting selectivity for the TPalpha isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC50 was similar to that of the TPalpha isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TPalpha isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.