We had previously isolated several endogenous MuLV proviruses from BALB/c and AKR/J mouse DNAS. Molecular and biochemical characterization of the cloned endogenous MuLV DNAs indicated that about 50% were related to known MuLV proviruses (Class I). Restriction enzyme and nucleotide sequence analysis indicated that the Class I endogenous MuLV DNAs could be distinguished from known infectious MuLV proviruses due to the presence of a transposon-like 190 bp cellular insert in their LTRs and unique restriction sites. Furthermore, the env sequences associated with the majority of such endogenous MuLV DNAs were similar to those present in recombinant MCF MuLVs. In fact, one cloned endogenous AKR MuLV DNA, designated as A-12, was almost identical in sequence of its 5' env region with leukemogenic MCF-MuLV env genes. We have determined the genomic location of the A-12 provirus to be on chromosome 13 or 18. Unlike other endogenous MuLV DNAs, the A-12 endogenous provirus was present in several inbred as well as wild mouse DNAs. Nucleotide sequence comparison of the LTR, 3' pol and env regions of leukemogenic (MCF-13) and non-leukemogenic (MCF-111A) MuLVs indicated a high degree of base homology in the env region. The LTR of MCF-13 was highly related to the LTR present in xenotropic MuLVs whereas, the LTR associated with MCF-111A was identical except for 1 bp with ecotropic MuLV proviral LTRs. The 3' pol region of MCF-13 contained a 12 bp sequence characteristic of leukemogenic MCF MuLVs. This was absent in MCF-111A MuLV DNA. The leukemogenic potential of in vitro constructed recombinant MuLVs was tested. These viruses contained 5' MCF-13 LTR, gag, pol and env sequences and 3' endogenous MuLV env and LTR sequences. Newborn AKR mice inoculated with such recombinant viruses developed thymic lymphomas in about 3 months. Tissue-specific expression of endogenous MCF-related mRNAs was detected in various AKR/N mouse tissues using P32-labeled MCF env-specific synthetic DNA probe. A 7.2 kb mRNA species was identified in the thymus which could be involved in the generation of recombinant thymotropic MCF viruses.