The parathyroid hormone (PTH) 2 receptor is potently activated by tuberoinfundibular peptide (TIP39). Rat and human PTH2 receptors differ considerably in their PTH responsiveness. PTH weakly stimulates cAMP accumulation via the rat receptor, and here we show it did not detectably increase intracellular calcium ([Ca2+]i) and bound with low affinity (450 nM). For the human PTH2 receptor PTH was a full agonist for increasing cAMP, a partial agonist for increasing [Ca2+]i, and bound with high affinity (18 nM). In addition, the antagonists PTH(7-34) and TIP(7-39) bound with 10- to 49-fold lower affinity to the rat receptor. We investigated the molecular basis of differential PTH and antagonist interaction with human and rat PTH2 receptors by using chimeric human/rat PTH2 receptors. PTH cAMP-signaling efficacy (Emax) was determined by extracellular loop (EL) 1 and a region including EL2 and EL3. The N-terminal domain determined PTH binding selectivity at the inactive receptor state. Multiple regions throughout the receptor are required for the PTH-PTH2 receptor complex to adopt a high-affinity active state: inserting the rat receptor's N-terminal domain, EL1 or EL2/3, into the human receptor increased PTH's EC50 and reciprocal exchanges did not reduce EC50. This suggests the global receptor conformation prevents the rat receptor from adopting a high-affinity state when in complex with PTH. N-terminal ligand truncation, producing the antagonists PTH(7-34) and TIP(7-39), altered ligand interaction with the membrane-embedded domain of the receptor, eliminating EL2/3 as a specificity determinant and lowering binding affinity. These insights should contribute to the development of a high-affinity PTH2 receptor antagonist, for investigating the receptor's physiological role.