The biologic activities of human parathyroid hormone-related protein [hPTHrP(1–34] and bovine PTH [bPTH(1-34)] are remarkably similar despite marked sequence divergence in their primary binding domain, residues 25–34. Chicken PTHrP (cPTHrP) is identical to hPTHrP through residue 21. However, in the 25–34 region, cPTHrP displays three fewer basic residues than hPTHrP and contains five residues not present in any other member of the PTH/PTHrP family. To assess the biologic consequences of these structural differences, we compared the activities of synthetic [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 with those of bPTH(1–34) in avian systems (chicken renal plasma membranes and 19 day chick embryonic bone cells) and mammalian systems [canine renal plasma membranes and rat osteosarcoma cells (UMR-106-H5)]. In both avian and mammalian systems the binding affinity of [36Tyr]cPTHrP(1–36)NH2 (0.8–3.4 nM) was approximately one-half that of hPTHrP(1–34)NH2 (0.4–1.1 nM). The potencies of [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 for activation of adenylate cyclase were similar in canine renal membranes (5.2 and 6.7 nM) and chick bone cells (1.0 nM). In UMR-106 cells and chicken renal membranes the potency of [36Tyr[cPTHrP(1–36)NH2 for activation of adenylate cyclase was about one-half that of [36Tyr]hPTHrP(1–36)NH2. Binding of 125I-[36Tyr]cPTHrP(1–36)NH2 to chick bone cells and chicken renal membranes was completely displaced by bPTH(1–34) and hPTHrP(1–34)NH2: thus there was no evidence for a distinct chicken PTHrP receptor. In general, [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 activated adenylate cyclase similarly despite their sequence differences in the 25–32 region. This suggests that basic residues in the 25–32 region are not required for the peptide to assume a biologically active conformation at the receptor. In cross-linking studies, both 125I-hPTHrP(1–34)NH2 and 125I-[36Tyr]cPTHrP(1–36)NH2 labeled a major 85 kD PTH/PTHrP receptor component in canine renal plasma membranes. 125I-hPTHrP(1–34)NH2 also labeled a ≤14 kD receptor fragment, whereas 125I-[36Tyr]cPTHrP(1–36)NH2 did not. The present results suggest that retained sequence features in the 25–32 region may be critical determinants of receptor binding and that sequence differences in this region alter the sites of interaction of PTHrP peptides with the PTH/PTHrP receptor.