Hypochondroplasia is a genetic disorder of disproportionate short stature. Linkage analysis provisionally placed hypochondroplasia in the chromosome 4p 16.3 region, a location to which the FGFR3 gene has been mapped. The genotyping of a three-generation family showed no recombinants between the hypochondroplasia phenotype and three highly polymorphic markers flanking the FGFR3 gene. Mutation analysis was performed by RT-PCR and direct sequencing. Primers covering most of the coding sequence of the FGFR3 gene were used for RT-PCR of FGFR3 mRNA and PCR amplification of genomic DNA. A C-->A transversion was detected in nucleotide 1659 predicting an N540K substitution in exon 11 which encodes part of the TK1 domain. The same mutation was found in an individual suspected to be an achondroplasia/hypochondroplasia compound phenotype and affected individuals from three other unrelated families. A second mutation, a C-->G transversion, also in nucleotide 1659 was detected in all affected individuals of another family. The latter also predicts an N540K substitution. These findings establish that a common mutation in the FGFR3 gene underlies hypochondroplasia.