Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to a common PTH/PTHrP receptor. To explore structure-function relations in these ligands, we synthesized and functionally evaluated PTH-PTHrP hybrid peptides in which the homologous 1-14 portions were exchanged. Hybrid-2, PTH-(1-14)-PTHrP-(15-34)NH2, bound to LLC-PK1 cells expressing the cloned rat PTH/PTHrP receptor with high affinity (IC50 approximately equal to 7 nM). In contrast, hybrid-1, PTHrP-(1-14)-PTH-(15-34)NH2, bound with much weaker affinity (IC50 approximately equal to 8,700 nM). Thus, the 1-14 region of PTHrP is incompatible with the 15-34 region of PTH. The carboxyl-terminal incompatibility site was identified as residues 19-21 (Glu-Arg-Val in PTH and Arg-Arg-Arg in PTHrP); extending the amino-terminal PTHrP sequence to residue 21 but not to 18 cured the hybrid's binding defect. The amino-terminal incompatibility site was identified as position 5 (Ile in PTH and His in PTHrP), because Ile5-hybrid-1 bound with high affinity (IC50 approximately equal to 20 nM). The importance of these identified residues in the native ligands was established by evaluating the effects of substitutions at these sites in a series of PTH and PTHrP analog peptides. Overall, the results are consistent with the hypothesis that, in both PTH and PTHrP, the 1-14 and 15-34 domains interact when binding to the receptor and that residues 5, 19, and 21 contribute either directly or indirectly to this interaction.