Residue 19 of the parathyroid hormone (PTH) has been shown to play an important role in both binding to and activation of the PTH receptor; specifically, Arg(19)-containing analogues have improved biological function over similar Glu(19) peptides [Shimizu et al. (2002) Biochemistry 41, 13224-13233]. Additionally the juxtamembrane portion of the receptor is involved in the different biological responses. Here, we determine the conformational preferences of PTH analogues to provide a structural basis for their biological actions. On the basis of circular dichroism results, the Arg(19) --> Glu(19) mutations within the context of both PTH(1-20) and PTH(1-34) analogues lead to increases in helix content, ranging from a 8-15% increase. High-resolution structures as determined by (1)H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the difference between Arg(19) and Glu(19)-PTH(1-20), particularly with the extent and stability of the C-terminal helix. The Arg(19)-containing analogue has a well defined, stable alpha-helix from Ser(4)-Arg(19), while the Glu(19) analogue is less ordered at the C-terminus. On the basis of these observations, we propose that position 19 of PTH(1-20) must be alpha-helical for optimal interaction with the juxtamembrane portion of the receptor. This mode of binding extends the current view of PTH binding (indeed ligand binding for all class B GPCRs), which invokes a bihelical ligand with the C-terminus of the ligand interacting with the N-terminus of the receptor (responsible for binding) and the N-terminus of the ligand interacting with the seven-helical bundle (leading to receptor activation).