The endogenous mouse mammary tumor virus (MMTV) proviral copies in two genetically dissimilar mouse strains, STS/A, a European mouse strain, and BALB/c, were characterized. STS/A carries the same four MMTV proviral copies as GR.Mtv-2-; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mtv-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mtv-2- is derived from GR; they differ in the locus Mtv-2 that contains one MMTV provirus. Expression of this Mtv-2 endogenous MMTV provirus is directly linked to mammary tumorigenesis in GR. MMTV proviral loci were studied using restriction enzyme analysis and the Southern transfer procedure using liver DNAs from recombinant inbred strains between BALB/c and STS/A. All segregating MMTV-specific EcoRI fragments were identified to MMTV proviral loci and most of these were localized by studying the cosegregation of the Mtv units and known chromosomal markers. Since STS/A, GR.Mtv-2-, and GR are highly related, the five complete endogenous MMTV proviruses of GR were located on the following chromosomes: Mtv-2 on chromosome 18, Mtv-3 on 11, Mtv-19 on 1, Mtv-20 on 4, whereas Mtv-8 has tentatively been located on chromosome 18 by Callahan et al. (R. Callahan, D. Gallahan, and Ch. Kozak (1984), J. Virol. 49, 1005-1008). GR and GR.Mtv-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A.