Using two separate methods, we have determined that all six potential sites for N-linked glycosylation on the rat lutropin/choriogonadotropin receptor (rLHR) contain carbohydrates. The functional roles of the carbohydrates were analyzed initially through the use of two nonglycosylated receptor mutants rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77,152,269,277,291)Q;T(175)A). Although Western blot analyses demonstrated both mutant receptors to be stably expressed, little or no hCG binding activity could be detected in detergent solubilized extracts of 293 cells expressing either nonglycosylated LHR mutant. Although this loss of hCG binding was concluded to be due to misfolding, it was unknown whether this misfolding was due to the absence of carbohydrates or to the multiple amino acid substitutions that had been introduced into the polypeptide. To differentiate between these possibilities, hCG binding assays were performed with nonglycosylated receptors obtained after tunicamycin treatment of cells expressing the wild-type rLHR. Even though these wild-type receptors were confirmed to be devoid of all N-linked carbohydrates by Western blots, they were found to bind hCG with a normal high affinity. In addition, tunicamycin-derived, nonglycosylated LHRs were present at the cell surface and exhibited a phenotype consistent with mature receptors due to their capability to mediate hCG-stimulated cAMP production as well as bind oLH with high affinity. These results indicate that the loss of high affinity hormone binding by rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77,152,269,277,291)Q;T(175)A) is simply due to the collective amino acid substitutions rather than to the absence of carbohydrates. Therefore, N-linked carbohydrates are not absolutely required for the proper folding of the rLHR into a mature receptor capable of binding hormone and signaling. These results are in marked contrast to the follitropin receptor (FSHR), a very similar receptor which has been shown to strictly require N-linked carbohydrates for folding of the nascent protein.