Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporated into the shortest fragment of hPTH with recognized efficacy in the OVX rat model of osteoporosis, hPTH(1-31)NH2 (1). Cyclo(Lys(18)-Asp(22))[Ala(1),Nle(8),Lys(18),Asp(22),Leu(27)]hPTH( 1-31)NH2 (2) is a potent agonist of the PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulate adenylyl cyclase activity (EC50 = 0.29 nM). A second analogue, which constrains the entire C-terminal receptor binding domain, bicyclo(Lys(18)-Asp(22),Lys(26)-Asp(30))[Ala(1),Nle(8),Lys(18),Asp(22),Leu(27)] hPTH(1-31)NH2 (6), is also shown to be a potent agonist (EC50 = 0.13 nM), thus providing further evidence for an extended helix as the relevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated into the analogue bicyclo(Lys(13)-Asp(17),Lys(18)-Asp(22)) [Ala(1),Me-8,Lys(18),Asp(17,22),Leu(27)]hPTH(1-31)NH2 (7) to evaluate the receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is also a highly potent agonist (EC50 = 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactive form of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporation of all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported, tricyclo(Lys(13)-Asp(17),Lys(18)-Asp(22),Lys(26)-Asp(30))[Ala(1),Nle(8),Asp(17,22),Leu(27)]hPTH(1-31)NH2 (9). Peptide 9 (EC50 = 0.14 nM) forces residues 13-30 into an extended helical conformation and is a more potent PTH receptor agonisr, than the parent linear hPTH(1-31)NH2 (1, EC50 = 4.7 nM). Comparative circular dichroism studies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1-12 are also likely to be helical in the bioactive conformation. Taken together, these results provide strong structural evidence that hPTH binds to its receptor in an extended helical conformation.