The RUNX3 gene is regarded as a tumor suppressor gene in many human solid tumors, and its inactivation is believed to be related with solid tumor carcinogenesis. As little information is available about the role of the RUNX3 gene in breast cancer, we investigated the relationship between the RUNX3 gene and breast cancer. We performed reverse transcriptase-polymerases chain reaction (RT-PCR), methylation specific PCR, and bicolor fluorescent in situ hybridization analysis in an effort to reveal related mechanisms. Forty breast tissue samples and 13 cell lines were used in this study. Eighty-five percent of breast cancer tissues showed downregulated RUNX3 gene expression, whereas it was downregulated in only 25% of normal breast tissues by RT-PCR assay. Sixty-seven percent of breast cancer cell lines showed downregulated RUNX3 expression, but the RUNX3 gene was not expressed in two normal breast cell lines. Hypermethylation was observed in 53% of breast cancer tissues and 57% of breast cancer cell lines. Hemizygous deletion was observed in 43% of breast cancer cell lines. Hypermethylation and/or hemizygous deletion was observed in 5 of 7 breast cancer cell lines, and the four of these five examined showed no RUNX3 gene expression. We suggest that various mechanisms, including methylation and hemizygous deletion, could contribute to RUNX3 gene inactivation.