BORIS (CTCFL) is the only known paralog of the versatile regulatory protein CTCF, a multifunctional DNA binding protein that mediates distinct gene regulatory functions involved in cell growth, differentiation, and apoptosis. Unlike CTCF, the expression of BORIS is normally restricted to specific cells in testes (the only cells where CTCF is not expressed), where it may play a role in reprogramming the methylation pattern of male germ line DNA. Frequent amplification of the 20q13.2 region, which contains the BORIS gene, and expression of BORIS transcripts in diverse human tumors and cell lines have led to the hypothesis that aberrant expression of BORIS may play a role in tumorigenesis by interfering with CTCF functions. However, recent studies using more quantitative methods indicate low frequency of BORIS expression in melanoma, ovarian, prostate, and bladder carcinomas. To investigate the relationship between chromosome 20q13 amplification and BORIS mRNA levels within breast cancer cell lines and tissues, we developed a quantitative RT-PCR assay to measure the levels of BORIS mRNA. Endpoint RT-PCR assays were also used to investigate the possible expression of alternatively spliced variants. Using multiple primer sets and controls, we found that neither mature BORIS transcripts nor spliced variants are commonly expressed at detectable levels in malignant breast cells or tissues, although endogenous BORIS transcripts can be induced in MCF-7 cells following 5-aza-2'-deoxycytidine treatment. In conclusion, in most breast cancer cells, endogenous BORIS is unlikely to be expressed at sufficient levels to interfere with CTCF functions. Thus it is improbable that aberrant BORIS expression plays a role in most human breast cancers.