Mouse mammary tumor virus (MMTV) has been related to human breast cancer (BC) in previous studies. Although suggestive sequence homology to MMTV has been described in BC DNA, the presence of human endogenous retroviruses (HERs) confounded these results. We have selected a 660-bp sequence of the MMTV env gene with very low homology to HER or to any other human or viral gene. We have searched for sequences homologous to it using the polymerase chain reaction. DNA was extracted from fresh or frozen tissues using primers and probes constructed to detect 660 bp; for paraffin-embedded tissues, we sought 250-bp sequences by similar methodology. The 660-bp sequence was detected in 121 (38.5%) of the 314 unselected BC samples, in cultured BC cells, in 2 (6.9%) of 29 breast fibroadenomas and in 2 (1.8%) of 107 breast specimens from reduction mammoplastias. The sequence was not found in normal tissues including breast, lymphocytes from BC patients, nor in other human cancers or cell lines. The 250-bp sequence was detected in 60 (39.7%) of the 151 BCs, and in 1 of 27 normal breast samples assayed from paraffin-embedded sections. Cloning and sequencing of the 660 bp and 250 bp demonstrated that they are 95-99% homologous to MMTV env gene, but not to the known HERs nor to other viral or human genes (< 18%). Southern blot analysis using labeled cloned sequences showed that the 660-bp sequences were present in low copy number as a 7-8-kb EcoRI fragment only in breast cancer samples and two breast cancer cell lines that were positive by PCR. These data indicate that 38-40% of human breast cancers contain gene sequences homologous to the MMTV env gene that are absent from other tumors and tissues. These MMTV env gene-like sequences may play a role in the etiology of a large proportion of human breast cancer.