The most extensively characterized thromboxane/prostaglandin endoperoxide (TP) receptors, from human platelets and rat vascular smooth muscle, exhibit thromboxane agonist [15-(1α,2β(5Z),3α-(1E,3S),4α)]-7-[3- hydroxy-4-(p-iodophenoxy)-1-butenyl-7-oxabicycloheptenoic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP having the higher affinity. We utilized this difference was found in the rank order of affinities for a series of thromboxane receptor ligands to bind to cloned human TP a versus rat TP, indicating that these represent species homologs, not distinct TP subtypes. Structural determinants for observed differences in I-BOP binding K(d) were localized by creating chimeric receptors with splice sites in transmembranes 1,2,4, or 7 were constructed and expressed in HEK293 cells for analysis of ligand binding properties. Substitution of any part except the carboxyl tail of the human TP into the rat TP resulted in a receptor with I-BOP binding affinity intermediate between the two. Analysis of chimeras in which only the extracellular amino terminus and a portion of transmembrane 1 were switched localized the determinant of high affinity I- BOP binding. Only when amino acid side chain at position 37, thus extending this group further into the putative I-BOP binding pocket, with compensatory shortening of side chains in spatially adjacent amino acids.