Three chemically-induced precancerous mammary hyperplasias, independently isolated in BALB/c mice, all contained mouse mammary tumor virus (MMTV) proviral DNA integrated into a common region in chromosomal DNA, designated Int-5 (Formerly Int-H, Gray et al., 1986). This site was cloned from a hyperplastic outgrowth (D2) into lambda phage. A 1.7 Kb Hind III DNA fragment, which flanks the 5' end of the MMTV insert, was generated from the cloned Int-5 region. This fragment was used as probe (IH-2) to localize Int-5 to mouse chromosome 9. The IH-2 sequence was highly conserved in DNA of several mammalian species including man, in three other widely divergent vertebrate phyla, and in C. elegans. The Int-5 region, containing 5.6 Kb 5' and 12.8 Kb 3' to the MMTV integration site in D2, was cloned in EMBL-3 from a BALB/c genomic DNA library. cDNA complementary to poly A+ lactating mammary gland RNA, annealed with Sst-1 fragments spanning most of the BALB/c Int-5 clone. The highest level of Int-5 specific poly A+ mRNA was detected in D2 tumor. Lactating mammary gland and D2 hyperplastic alveolar nodule contained 5-fold less Int-5 RNA while liver contained 8-fold less Int-5 RNA. Int-5 cDNA (IH-3) annealed with two RNA species of approximately 3.3 and 4.0 Kb. These data are consistent with the hypothesis that Int-5 contains an oncogene, different from any other previously described, involved in early events in some models of chemical carcinogenesis.