The spontaneous leukemias of AKR mice are caused by mink cell focus-forming (MCF) viruses. These viruses are generated by recombination between several endogenous murine retroviruses. The virological events leading to the generation of the leukemogenic agent were investigated by using an oligonucleotide specific for the U3 region of the leukemogenic virus and env-reactive oligonucleotide probes specific for the different classes of endogenous murine leukemia virus. It was shown that (i) the leukemogenic MCF virus is formed by recombination between at least three different endogenous sequences; (ii) the U3 donor for the leukemogenic virus is the inducible xenotropic virus Bxv-1; (iii) all spontaneous tumors contain viruses with duplicated enhancer regions in their long terminal repeats; (iv) enhancer duplication is a somatic event, since Bxv-1 contains only one copy; (v) the first recombinant virus detectable in mass populations of thymocytes by Southern hybridization analysis contains all structural features of the ultimate leukemogenic virus; and (vi) the multiple novel viruses in a given tumor represent progeny of the same unique recombination events. On the basis of these results, an analysis of the virological events leading to AKR thymomas is presented.