BACKGROUND Integration of retroviral DNA into a germ cell can result in a provirus that is transmitted vertically to the host's offspring. In humans, such endogenous retroviruses (HERVs) comprise >8% of the genome. The HERV-K(HML-2) proviruses consist of ~90 elements related to mouse mammary tumor virus, which causes breast cancer in mice. A subset of HERV-K(HML-2) proviruses has some or all genes intact, and even encodes functional proteins, though a replication competent copy has yet to be observed. More than 10% of HML-2 proviruses are human-specific, having integrated subsequent to the Homo-Pan divergence, and, of these, 11 are currently known to be polymorphic in integration site with variable frequencies among individuals. Increased expression of the most recent HML-2 proviruses has been observed in tissues and cell lines from several types of cancer, including breast cancer, for which expression may provide a meaningful marker of the disease. RESULTS In this study, we performed a case-control analysis to investigate the possible relationship between the genome-wide presence of individual polymorphic HML-2 proviruses with the occurrence of breast cancer. For this purpose, we screened 50 genomic DNA samples from individuals diagnosed with breast cancer or without history of the disease (n = 25 per group) utilizing a combination of locus-specific PCR screening, in silico analysis of HML-2 content within the reference human genome sequence, and high-resolution genomic hybridization in semi-dried agarose. By implementing this strategy, we were able to analyze the distribution of both annotated and previously undescribed polymorphic HML-2 proviruses within our sample set, and to assess their possible association with disease outcome. CONCLUSIONS In a case-control analysis of 50 humans with regard to breast cancer diagnosis, we found no significant difference in the prevalence of proviruses between groups, suggesting common polymorphic HML-2 proviruses are not associated with breast cancer. Our findings indicate a higher level of putatively novel HML-2 sites within the population, providing support for additional recent insertion events, implying ongoing, yet rare, activities. These findings do not rule out either the possibility of involvement of such proviruses in a subset of breast cancers, or their possible utility as tissue-specific markers of disease.