The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (ΔrFSHR) cDNAs were expressed in insect cells. The large deletion ΔrFSHRS9-N30 and a smaller deletion, ΔrFSHRS9-S18, did not bind ¹²⁵I-hFSH. However, ΔrFSHRK19-R29 and ΔrFSHRD300-F315 bound ¹²⁵I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants ΔrFSHR S9-N30 or ΔrFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although ¹²⁵I-hFSH binding to ΔrFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas ΔrFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to ΔrFSHR.