Insulin-like growth factor I (IGF-I), a 70-amino acid basic polypeptide, plays a fundamental role in postnatal mammalian growth as a major mediator through which growth hormone exerts its biological effects. We have recently identified two human IGF-I cDNAs which predict distinct peptide precursors of 153 and 195 amino acids. In the present study, both cDNAs were used to isolate and characterize the human IGF-I gene from genomic libraries. The IGF-I gene extends over at least 45 kilobase pairs and contains five exons interrupted by four introns. The DNA sequence of exons 1 through 4 encodes the 195-amino acid precursor, while exons 1, 2, 3, and 5 code for the 153-residue peptide, confirming the hypothesis that at least two IGF-I mRNAs are generated by alternative RNA processing of the primary gene transcript. The structure of the IGF-I gene resembles that of its companion somatomedin, IGF-II, as judged by the analogous location of two introns and considerable nucleotide and amino acid sequence similarity, but appears more distantly related to other members of the insulin gene family. Restriction endonuclease polymorphisms in the IGF-I gene, which map near exon 5 as determined by Southern blot analysis, will be useful in defining the genetics of familial growth failure.