The insulin receptor substrates (IRSs) are key proteins in signal transduction from the insulin receptor. Recently, we discovered a fourth member of this family, designated IRS-4, cloned its complementary DNA from the human embryonic kidney 293 cell line, and characterized its signaling properties in this cell line. As part of an investigation of the physiological role of this IRS, we have now cloned the mouse IRS-4 gene and determined its tissue expression and chromosomal location. The coding region of the mouse IRS-4 gene contains no introns, and in this regard is the same as that of the genes for IRS-1 and -2. The predicted amino acid sequence of mouse IRS-4 is highly homologous with that of human IRS-4; the pleckstrin homology domain, the phosphotyrosine-binding domain, and the tyrosine phosphorylation motifs are especially well conserved. The tissue distribution of IRS-4 in the mouse was determined by analysis for the expression of its messenger RNA by RT-PCR and for the protein itself by immunoprecipitation and immunoblotting. The messenger RNA was detected in skeletal muscle, brain, heart, kidney, and liver, but the protein itself was not detected in any tissue. These results indicate that IRS-4 is a very rare protein. The chromosomal locations of the mouse IRS-4 and IRS-3 genes were determined by interspecific back-cross analysis and were found to be on chromosomes X and 5, respectively. As the mouse genes for IRS-1 and -2 are on chromosomes 1 and 8, respectively, each IRS gene resides on a different chromosome.