Polypeptides that activate the parathyroid hormone receptor-1 (PTHR1) are important in human physiology and medicine. Most previous studies of peptide binding to this receptor have involved the displacement of a radiolabeled ligand. We report a new assay format based on bioluminescence resonance energy transfer (BRET). Fusion of a NanoLuc luciferase (nLuc) unit to the N-terminus of the PTHR1 allows the direct detection of binding by an agonist peptide bearing a tetramethylrhodamine (TMR) unit. Affinity measurements from the BRET assay align well with results previously obtained via radioligand displacement. The BRET assay offers substantial operational benefits relative to affinity measurements involving radioactive compounds. The convenience of the new assay allowed us to explore several questions raised by earlier reports. For example, we show that although the first two residues of PTH(1-34) (the drug teriparatide) are critical for PTHR1 activation, these two residues contribute little or nothing to affinity. Comparisons among the well-studied agonists PTH(1-34), PTHrP(1-34), and "long-acting PTH" (LA-PTH) reveal that the high affinity of LA-PTH arises largely from a diminished rate constant for dissociation relative to the other two. A D-peptide recently reported to be comparable to PTH(1-34) as an agonist of the PTHR1 was found not to bind detectably to the receptor and to be a very weak agonist.