Insulin-like growth factor binding proteins (IGFBPs) constitute a family of at least six secreted proteins that bind insulin-like growth factors I and II (IGF-I and -II) and are capable of modifying IGF actions on target cells. We previously have purified an approximately 29-kDa IGFBP that is secreted by myoblasts during their terminal differentiation, have identified the protein as mouse IGFBP-5, and have cloned its cDNA (James et al., 1993). In this study, we have characterized the mouse IGFBP-5 gene, established its pattern of expression in the adult mouse, and defined its chromosomal location. The 17-kb gene was isolated on overlapping cosmid and lambda clones, and the four exons encoding the 5914-bp mRNA were sequenced. The 5' end of the gene was mapped by solution-hybridization ribonuclease protection assay to two discrete sites in exon 1 that were separated by 21 bp. The relative use of each transcription start site was found to vary among different mouse tissues. By interspecies backcross mapping using progeny derived from matings of [(C57BL/6J X Mus spretus)F1 x C57BL/6J] mice, the IGFBP-5 gene was localized to the proximal region of chromosome 1 in tight linkage with fibronectin 1 (0 recombinants in 168 mice analyzed). Since this part of chromosome 1 shares homology with human chromosome 2q, and since fibronectin has been mapped to 2q34-q36, it is likely that human IGFBP-5 will reside on 2q as well. Characterization of the mouse IGFBP-5 gene now provides a starting point for studying the roles and regulation of this protein in development.