Novel strategies for early detection of breast cancer, the most common and second most lethal cancer in women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including breast cancer. Detecting such epigenetic changes in DNA derived not only from tumor tissue, but also from bodily fluids, may be a promising target for the molecular analysis of cancer. In this study we examined serum, a readily accessible bodily fluid known to contain neoplastic DNA, from individuals with breast carcinoma. Using sensitive methylation-specific polymerase chain reaction, we searched for aberrant promoter hypermethylation of two normally nonmethylated genes: RAS association domain family member 1A (RASSF1A) and death-associated protein kinase 1 (DAPK1) in 26 patients with breast cancer, 16 patients with benign breast diseases, and 12 age-matched healthy controls. Hypermethylation of at least one gene was detected in 25/26 (96%) cancer patients, in 7/16 (43%) cases with benign breast diseases, and only 1/12 (8%) control subjects. Furthermore, methylation of both genes was found to be associated with ductal type of breast carcinoma. RASSF1A was hypermethylated in 18/26 cases (69%) and DAPK1 in 23/26 (88%). However, DAPK1 promoter methylation was more pronounced, as 12/23 DAPK1 methylated cases (52%) were strongly methylated (>75%) compared to the weaker methylation of RASSF1A (none of the cases with methylation at the level of >75%). These findings, if confirmed in studies of extended cohorts, may lead to useful clinical application in early diagnosis of breast cancer and better management of the neoplastic disease.