To identify determinants in the rat PTH receptor critical for binding the agonist peptide, PTH-(1-34), we systematically replaced 12 segments (5-33 residues) of the receptor's extracellular surface with the corresponding segments of the homologous rat secretin receptor and screened the resulting mutants in COS-7 cells for altered PTH-(1-34) binding properties. Surface expression of mutant receptors was assessed by the binding of monoclonal antibody 12CA5 to the epitope (HA)-tagged receptors. Of the nine well expressed and therefore informative receptor mutants, four bound radiolabeled PTH-(1-34) at levels that were proportional to the corresponding levels of surface expression, whereas five mutants bound [125I]PTH-(1-34) to levels that were lower than predicted from the cell surface expression levels. These five mutations occurred at the extracellular (EC) end of transmembrane domain 1, the carboxy-terminal portion of the first EC loop, the second EC loop, and the third EC loop. We selected for further fine structure analysis the third EC loop; two specific residues, Trp-437 and Gln-440, were identified at which mutations caused 9- to 16-fold reductions in PTH-(1-34)-binding affinity. The same mutations had little or no effect on the binding affinity of PTH-(3-34). This study provides new information on the location of PTH receptor regions important for high affinity agonist binding and identifies two residues in the third extracellular loop which may contribute to interactions involving the hormone's critical amino terminus.