The highly specific ligand [125I]bovine (b) PTH-(1-34) and a chemical cross-linking technique were used to explore structural features of the canine renal cortical PTH receptor. Membranes isolated under conditions designed to inhibit endogenous proteolysis displayed a major 85K labeled PTH receptor moiety on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked receptors were solubilized with Lubrol-PX and partially purified by affinity chromatography on wheat germ agglutinin-agarose, and their hydrodynamic properties were assessed [Stokes radius = 7.3 +/- 0.1 nm; sedimentation coefficient = 6.4 +/- 0.2S; partial specific volume = 0.758 +/- 0.01 ml/g; frictional coefficient = 1.68 +/- 0.04; mol wt (Mr) = 216,000 +/- 14,000]. Corrections for detergent binding and for the presence of carbohydrate yielded an estimated Mr of 166,000 +/- 11,000 for the solubilized PTH receptor. Thus, the renal PTH receptor is oligomeric, with a Mr approximating that expected of a homodimer of 85K subunits. Peptide-mapping experiments revealed the presence within the 85K PTH receptor subunit of at least two major regions sensitive to proteolytic attack. Both elastase and an endogenous renal protease(s) cleaved the PTH receptor to a 70K form that is fully functional with respect to high affinity, guanyl nucleotide-sensitive PTH binding. Cleavage in a second domain by elastase, S. aureus V8 protease, or chymotrypsin generated a 50K labeled PTH receptor fragment. Cleavage at this second site was prevented by prior occupancy of the receptor with [125I]bPTH-(1-34), suggesting that this domain may be functionally important. Reduction of receptor disulfide bonds with dithiothreitol and beta-mercaptoethanol released a low Mr (less than or equal to 14K) labeled PTH receptor component, similar treatment of renal membranes abolished specific PTH binding, indicating that an intact disulfide bond(s) is essential for receptor function. These results provide new insights into the structural basis of PTH receptor function.