HES-3 is a cerebellar Purkinje cell-specific helix-loop-helix factor structurally related to the products of the Drosophila hairy and Enhancer of split genes. Here, we report the nucleotide sequence and chromosomal locus of the mouse Hes-3 gene. This gene consists of four exons and the exon-intron boundaries are well conserved when compared with those of the mouse Hes-1 and Drosophila hairy genes. Southern blot and interspecies backcross analyses show that the mouse Hes-3 gene is a single-copy gene and is located around position 80 on chromosome 4. Further analysis indicates that this locus is close to the Hes-5 locus, which is different from the Hes-1 locus (position 26 on chromosome 16). These results suggest that the Hes-3 and Hes-5 genes may be clustered on chromosome 4 while the Hes-1 gene is not.