There are several reported mutations of the GH-Receptor (GH-R) gene in subjects with complete or partial growth hormone insensitivity syndrome (GHIS) where the relationship between mutation and phenotype is obscure. We have used Southern blotting, SSCP and DGGE screening and direct DNA sequencing to test the hypothesis that multiple mutations can be present in GH-R alleles from subjects with GHIS. Two Oriental Jewish siblings with GHIS were found to be homozygous for a GH-R allele that contained three departures from the normal sequence. Genomic deletion of exon 3, in this and 3 other families with GHIS, did not explain the disease because previous studies of expressed receptors showed no effect of exclusion of exon 3 on GH binding. An R211H mutation in exon 7 did not explain the disease because the expressed GHBP with the mutation had near normal GH binding (Goddard et al, NEJM 1995;333:1093-1098). The third and decisive mutation changed codon 4 in exon 2 from tryptophan to a translational stop codon. Thus, the protein product of this multiply mutated GHIS allele would contain only 3 amino acids and the downstream mutations can be considered irrelevant. We suggest that complete characterization of GHIS alleles is necessary. This is simple, as in our subjects, parental consanguinity implies homozygosity by descent. The presence of more than one mutation in a single GH-R allele will be equally important, but more difficult to demonstrate in a more diverse population sample. Supported by NIH Grants DK46312 and DK07298-13.