Mechanisms of ligand binding to the PTH(PTH)/PTH-related protein receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, ¹²⁵I-[Aib^1,3,Nle⁸,Gln ¹⁰,homoarginine¹¹,Ala¹²,Trp¹⁴,Tyr¹⁵]rPTH(1-15)NH₂ (¹²⁵I-[Aib ^1,3,M]PTH (1-15)) was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed ¹²⁵I-PTH (1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of ¹²⁵I-[Aib^1,3,M]PTH (1-15) to the wild-type PTHR was strongly (90%) inhibited by GTPS, whereas the binding of ¹²⁵I-PTH (1-34) was only mildly (25%) inhibited by GTPS. Of these two radioligands, only ¹²⁵I-[Aib^1,3,M]PTH (1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPS. Binding of ¹²⁵I-[Aib^1,3,M]PTH (1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (20%) inhibited by GTPS, as was the binding of ¹²⁵I-PTH (1-34). In membranes prepared from cells lacking G_S via "knock-out" mutation of Gnas, no binding of ¹²⁵I-[Aib^1,3,M]PTH (1-15) was observed, but binding of ¹²⁵I-[Aib^1,3,M]PTH (1-15) was recovered by virally transducing the cells to heterologously express G_S. ¹²⁵I-PTH (1-34) bound to the membranes with or without G_S. The overall findings confirm the hypothesis that ¹²⁵I-[Aib^1,3,M]PTH (1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to G_S-containing heterotrimeric G proteins, whereas the binding of ¹²⁵I-PTH (1-34) can occur in the absence of such coupling. Thus, ¹²⁵I-[Aib^1,3,M]PTH (1-15) appears to function as a selective probe of G_S-coupled, active-state PTHR conformations.