We have isolated and characterized overlapping clones from phage lambda and cosmid human genomic libraries that predict the entire structure of the gene encoding the precursor to human growth hormone-releasing factor. The gene includes five exons spanning 10 kilobase pairs of human genomic DNA. There appears to be a segregation of distinct functional regions of the GRF precursor and its mRNA into the five exons of the gene. The DNA sequences of all exons, intron/exon boundaries, and 5' and 3' flanking regions are presented. Dot-blot analysis of DNA from high resolution dual-laser-sorted human chromosomes indicates that the single-copy growth hormone-releasing factor gene is located on human chromosome 20.