Laron syndrome is an autosomal recessive condition characterized by resistance to growth hormone. We sought to determine the molecular basis of this condition in an Ecuadorean population with a high incidence of affected individuals. Growth hormone receptor gene sequences from an obligate heterozygote were amplified by the polymerase chain reaction and screened for mutations using denaturing gradient gel electrophoresis. Only exon 6 revealed homo- and heteroduplexes on denaturing gradient gels. Sequencing revealed a substitution of guanine for adenine in the third position of codon 180 that did not change the amino acid encoded. Sequencing of the exon 6-exon 7 splice junction from RNA-polymerase chain reaction amplified cellular RNA of an affected individual revealed that the substitution activates a 5' splice site 24 nucleotides upstream from the normal exon 6-intron 6 boundary. Splicing in two probands' lymphoblasts occurred virtually exclusively at the abnormal 5' splice site created by the codon 180 substitution. Exon 6 sequences from 38 patients and 47 relatives were amplified and analyzed by sequencing or dot-blot hybridization with allele-specific oligonucleotides. The substitution was detected in 74 of 76 Laron syndrome patients' GH-receptor alleles. All 26 parents and 12 of 21 unaffected siblings were heterozygous for this mutation. It was absent in 61 unrelated unaffected control individuals. We conclude that the codon 180 nucleotide substitution probably causes Laron syndrome as translation of the observed, abnormally spliced growth hormone receptor transcript would lead to the synthesis of a receptor protein with an 8 amino acid deletion from the extracellular domain.