We have constructed a 64-cM molecular genetic linkage map of mouse chromosome 4 using interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. Several proto-oncogenes and common sites of viral integration have been assigned regional locations on chromosome 4 including Mos, Lyn, Jun, Lmyc, Lck, Fgr, and Dsi-1. Additional loci mapped in this study to chromosome 4 were Tsha, Mup-1, Rrm2-ps1, Ifa, and Anf. A comparison of our mapping data with inbred strain mapping data did not show any evidence for inversions or deletions on chromosome 4. New regions of synteny were defined between mouse chromosome 4 and human chromosomes 1 and 8; a region of homology was found between mouse chromosome 4 and human chromosome 6. This linkage map will provide a framework for identifying homologous genes in mice and humans that may be involved in various disease processes.