The DNA-PKcs gene encodes the 465-kDa catalytic subunit of DNA-dependent protein kinase (DNA-PK), which associates with heterodimeric autoantigens Ku70 and Ku80 and exhibits protein kinase activity depending on DNA double-strand breaks. The gene is also responsible for the aberration in severe combined immune deficiency (SCID) mice, which exhibit a high sensitivity to ionizing radiation and abnormal DNA rearrangement of immunoglobulin and T cell receptor genes. There is further evidence that DNA-PKcs phosphorylates various proteins involved in DNA replication, transcription, repair, and recombination. Nevertheless the structure/function relationship in this huge molecule is virtually unknown. We determined the exons and introns of the murine DNA-PKcs gene by the long-distance polymerase chain reaction method. The murine DNA-PKcs gene consists of 86 exons distributed in a region of more than 250 kb. The average size of the exons is 140 bp. All the splicing sites conform to the GT/AG rule. The SCID mutation site (Tyr4046) has been identified in exon 85. The genomic structure of the DNA-PKcs gene provides clues for the study of various functional domains in this macromolecule.