Approximately 50% of tumors induced by mouse mammary tumor virus (MMTV) contain an acquired provirus within a limited region of chromosomal DNA, termed int-2. We have extended our previous characterization of this locus and have mapped provirus integration sites in 21 independent tumors. Although integration occurs at multiple sites, proviruses within int-2 are distributed into two oppositely oriented groups whose transcription is directed away from a central domain. Provirus insertion in int-2 is accompanied by expression of RNA derived, at least in part, from this central domain. Since the RNA is not detected in normal mammary tissue, we conclude that MMTV integration activates the expression of a cellular gene within int-2 and that this event may contribute to tumorigenesis.