The Mlvi-1/mis-1/pvt-1 locus, located approximately 270 kilobase pairs 3' of the c-myc protooncogene, was originally discovered as a common region of provirus integration in Moloney murine leukemia virus-induced rat T-cell lymphomas. The same locus was shown subsequently to be coamplified with c-myc and to be involved in chromosomal translocations in a variety of human and animal neoplasms. Provirus integration in Mlvi-1 in Moloney murine leukemia virus-induced rat T-cell lymphomas activates the c-myc protooncogene. The studies reported here were aimed to determine whether, in addition to the activation of c-myc, provirus integration affected the expression of other neighboring genes. Provirus integration was shown to occur in three clusters separated by regions of uninterrupted DNA. The proviruses in all three clusters had integrated in a single-transcriptional orientation, and they appeared intact. Systematic hybridization of Mlvi-1 clones to rat, mouse, and human genomic DNA revealed three patches of evolutionarily conserved sequences. Two of them were mapped in regions targeted by the provirus, and the third was mapped immediately 5' to the provirus clusters. A probe derived from the conserved sequences 5' of the integrated proviruses detected a tumor-specific RNA transcript in tumors carrying a provirus in Mlvi-1 or in the neighboring Mlvi-4 and c-myc loci. The highest level of RNA transcript expression, however, was seen in a CD4+ CD8+ tumor cell line that was not carrying a provirus in this region. We conclude that provirus insertion in this region activates both c-myc and another gene that is located in the immediate vicinity of the integrated Mlvi-1 proviruses and may be developmentally regulated in T cells.