The genome of a recombinant murine leukemia virus capable of inducing focal areas of morphological alteration in mink lung fibroblasts was studied by heteroduplex analysis. The dual-tropic recombinant virus was isolated from a thymoma cell line (Th16.3) and is referred to as BALB/Moloney mink cell focus-inducing virus (BALB/Mo-MCF virus). The nucleic acid sequences of RNA from virions obtained from either a thymoma cell line (Th16.3) or a clonal isolate (BALB/Mo-MCF81) were compared with the genomes of ecotropic and xenotropic viruses. The following inferences were drawn (i) A single nonhomologous region (substitution loop alpha) of about 0.7 kilobase was observed in a heteroduplex formed between Moloney murine leukemia virus complementary DNA (cDNA) and BALB/MoMCF81 RNA. This nonhomology region was mapped between 1.71 and 2.40 kilobases from the 3' end of the genome. (ii) The predominant class of heteroduplexes formed between virion RNA obtained from the thymoma cell line (Th16.3) and Moloney murine leukemia virus cDNA showed a substitution loop similar to that observed with the RNA obtained from a cloned isolate, BALB/Mo-MCF81. However, there were other molecules with additional regions of nonhomology. (iii) Heteroduplexes formed between NZB xenotropic RNA and ecotropic Moloney murine leukemia virus cDNA exhibited four major nonhomology regions extending 0.75 to 1.46, 2.0 to 2.8, 3.6 to 4.3, and 7.4 to 7.9 kilobases from the 3' end of the genome. (iv) The MCF-specific substitution loop alpha (1.71 to 2.40 kilobases) appeared as a duplex region when NZB xenotropic RNA was hybridized to cDNA transcripts synthesized by virions obtained from thymoma cell line Th16.3. The position of the other substitution loops observed in a heteroduplex formed between NZB xenotropic RNA and Moloney murine leukemia virus cDNA was not affected. (v) Heteroduplexes formed between xenotropic BALB virus 2 cDNA and NZB xenotropic RNA demonstrated a large degree of nucleic acid sequence homology. Of the 29 heteroduplexes examined, 24 appeared to be homoduplexes, and in the remaining 5 heteroduplexes only one region of nonhomology located between 3.2 and 3.8 kilobases from the 3' end of the genome could be identified. Hybridization of BALB virus 2 xenotropic RNA to NZB xenotropic cDNA followed by digestion with single-strand-specific nuclease S1 showed an 80% sequence homology.