Isolated GH deficiency (IGHD) is familial in 5-30% of cases. The majority of patients have the type IB form, characterized by autosomal recessive transmission, low but measurable serum concentrations of GH, and responsiveness to exogenous GH therapy. Unique mutations in the gene encoding the GHRH receptor (GHRHR) have previously been described in 2 kindreds with IGHD IB. However, the prevalence of GHRHR mutations in patients with IGHD IB is unknown. We analyzed 30 families with IGHD IB in which more than 1 member was affected. Linkage analysis was performed in 28 of the families, and in 3 families sibling pair analysis indicated linkage to the GHRHR gene locus. These 3 families as well as 2 families in which linkage analysis was not performed were screened for mutations in the 13 coding exons, the intron-exon boundaries, and 327 bases of the promoter of the GHRHR gene. We identified novel GHRHR missense mutations in 2 of the 3 kindreds with informative linkage and in 1 family in which linkage had not been performed. In 1 family affected members were homozygous for a mutation in codon 144 that replaces leucine with histidine (L144H). Affected subjects in a second family were compound heterozygotes, carrying both the L144H mutation and a second mutation in codon 242 that replaces phenylalanine with cysteine. Affected subjects in a third family were homozygous for a mutation that replaces alanine at codon 222 with glutamic acid. All 3 mutations segregated with the IGHD phenotype. All 3 mutant receptors were expressed in CHO cells, and each failed to show a cAMP response after treatment of the cells with GHRH. These results demonstrate that missense mutations in the GHRHR gene are a cause of IGHD IB, and that defects in the GHRHR gene may be a more common cause of GH deficiency than previously suspected.