The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.