The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.