Abstract: Numerous mutated genes have been shown to be involved in breast cancer. The p53, a well-known tumor suppressor gene, is the most commonly mutated gene that plays an important role in directing cells with DNA damage into apoptosis. On the other hand, Estrogen Receptor (ER), an important prognostic factor is differentially expressed in breast cancer cells. Therefore, we decided to study the p53 gene and its protein in MCF7, T47D and MDA-MB-468 breast cancer cell lines with different ER status following exposure to Adriamycin (ADR). Cytotoxicity of ADR on these cell lines was determined using MTT assay. The mRNA and protein levels were also analyzed in cell lines using RT-PCR and immunocytochemistry (ICC) assays, respectively. ADR cytotoxicity was highest on ER negative MDA-MB-468 cells and lowest on ER positive MCF7 cells. The p53 mRNA level was highest before or after treatment with ADR in MDA-MB-468 and lowest in T47D cells. It is noteworthy to mention that the p53 mRNA level slightly increased in T47D cells but decreased in other two cell lines after ADR treatment. Interestingly, higher level of p53 protein expression was detected after ADR exposure in all three cell lines. In conclusion, these three cell lines with different ER status showed differential molecular responses to Adriamycin that is important in tumor-targeted cancer therapy.