Mouse chromosome 12 encodes the heavy chains of immunoglobulins (Igh), a family of T cell surface molecules, and a tumor antigen that may be homologous to immunoglobulins. To refine and extend the genetic map of this chromosome, a procedure has been developed to isolate chromosome 12-specific DNA fragments from a somatic cell hybrid carrying the chromosome on a Chinese hamster background. Five fragments have been isolated and characterized in detail. All are polymorphic, defining loci D12-1, 2, 3, 4, and 5. Using recombinant inbred mouse strains, a tentative linkage map of chromosome 12 has been worked out that incorporates these markers, the c-fos oncogene, Igh, and Pre-1/alpha 1 antitrypsin. This strategy should be applicable to any mouse chromosome or chromosomal region that can be isolated in a somatic cell hybrid.