We have identified and characterized a novel human insulin-like growth factor I (IGF-I) precursor from the transplantable T61 human breast cancer xenograft and from normal liver. The mRNA encoding this precursor contains a 5′-untranslated region that is 83% identical to the corresponding region of a previously described variant rat IGF-I. The nucleotide sequence of the cloned cDNA predicts an IGF-IA protein precursor of 137 amino acids, including a 32 residue signal peptide, 70 amino acid IGF-I, and a 35 residue COOH-terminal extension or E peptide. The exon encoding this variant maps in the genome between IGF-I exons 1 and 2, in a similar location to the homologous rat exon 1a. The rat and human exons 1a are 59% identical over 1443 nucleotides, with DNA sequence conservation occurring in a mosaic pattern. Human IGF-I mRNAs encoding this novel exon are expressed in liver, T61 tumor cells, and in an ovarian carcinoma cell line, NIH OVCAR3. These studies demonstrate that as in the rat, the human IGF-I gene contains six exons that are variably processed into multiple IGF-I mRNAs. The mechanisms responsible for generating different IGF-I mRNAs thus appear to be conserved among mammalian species.