We have developed an approach for genetic analysis of the murine H-2 complex that has broad general applicability to the study of eukaryotic genome organization. We have used a retroviral vector to introduce a selectable marker into the mouse genome close to the major histocompatibility complex (MHC). Chromosomal segments containing large portions of the MHC from these donor cells have been transferred both to hamster and monkey cell recipients. The procedure involved the following steps. First, a murine cell line was multiply infected with a defective recombinant murine leukemia virus that contains the neomycin-resistance gene (a gene that confers resistance to G418). In this way, the neomycin-resistance gene was introduced at multiple sites in the mouse genome. Second, metaphase chromosomes, prepared from this infected cell population, were transferred to hamster cell recipients. Third, two G418-resistant transferents were identified that expressed murine H-2 antigens on their cell surface. These transferents were shown to contain a large segment of the murine MHC (H-2K and I regions) by DNA hybridization. The neomycin-resistance gene and the mouse MHC genes must be physically linked in these cells since they could be cotransferred from the hamster cells to monkey cells. Fourth, the murine cell carrying the neomycin-resistance gene near the MHC was identified from the original donor cell population. This cell will serve as a useful source of chromosome fragments for analysis of larger portions of the MHC. This series of steps can serve as a paradigm for the first steps in a detailed genetic analysis of any specific region of a mammalian genome to which one or more genes have already been mapped.