We examined the GnRH gene structure in a family with familial central precocious puberty (eight members, four affected) and a family with idiopathic hypogonadotropic hypogonadism (eight members, three affected) using Southern blot analysis and sequencing of cloned polymerase chain reaction products. Genomic DNA samples were digested with restriction enzymes and hybridized to the human placental GnRH cDNA probe. BamHI digests revealed 6.5- and 2.7-kilobase (kb) bands; BglII, 6.0- and 4.0-kb bands; Ncol, 8.0- and 3.5-kb bands; Pstl, 4.2-kb, 2.8-kb, 1.3-kb and 950-basepair bands; XbaI, 6.5- and 5.0-kb bands. These sizes were the same as those found by this analysis in normal individuals. All family members with familial central precocious puberty or idiopathic hypogonadotropic hypogonadism showed the same size bands, except for one unaffected member of the family with idiopathic hypogonadotropic hypogonadism who had an additional band at 5.5 kb after digestion with NcoI, which is thought to be a rare polymorphism. Sequencing of exon 2 of the GnRH gene from these families, including the exon-intron borders, revealed a polymorphism in the signal sequence of GnRH that predicts an amino acid change from tryptophan (nucleotide sequence: TGG) to serine (TCG) at the -8 position of the GnRH preprohormone. Although this polymorphism did not cosegregate with the clinical disorder in either family, this novel polymorphism may prove useful in the evaluation of linkage to the GnRH gene in other families with pubertal disorders. No other nucleotide sequence abnormality was found in 1.2 kb of the 5' flanking region or the four exons and their splice sites.