Breast Cancer is the most common cancer in Iranian women. Breast tumors areclassified based on the estrogen receptor alpha (ER) expression status to ERnegative and ER positive tumors. Considering to the challenging nature of ERnegative tumors treatment, and their innate poor prognosis, clarification of themolecular mechanisms that control expression of ER is essential. Thisknowledge may enable us to restore responsiveness to endocrine therapies andprovide us new therapeutic options. Gene promoter methylation is one of themechanisms for gene silencing and because of its reversible nature; may be agood target for new therapeutic strategies. Thus we aimed to evaluate thecorrelation of ER promoter methylation with ER negative phenotype in 100breast tumors and its association to clinicopathological features and ERexpression in mRNA level. Also we mapped the CpG island in ER negative andER positive clones obtained from primary breast tumors to identify criticalCpGs. First ER promoter methylation was assessed with MSP technique intumors and was shown that ER negativity in tumors is correlated to ERmethylation. Also ER methylation was correlated to progesterone receptornegativity in tumors. There was no significant relationship between methylationand other clinicopathological features of the patients. ER gene expression studywith real time PCR showed that there is a significant correlation between ERmRNA level and methylation. Finally we scanned 55 CpG sites with bisulfitesequencing in 583 bp of ER promoter CpG island in 131 clones obtained frombreast tumors. Methylation patterns in studied ER negative primary tumors weredifferent from in vitro model for ER re-expression with epidrugs. Thus ERmethylation pattern in primary breast tumors is different from the current cellline model for epigenetic therapies and it reminds the importance of findingcellular models similar to tumors for evaluating epidrug therapies.