p53 1s a nuclear phosphoprotem whose function is classified as a tumor suppressor (1 ) Mutattons in the p53 gene are currently regarded as the most common genetic alteration in human cancer (2 ), including breast cancer (reviewed in ref. 3 ). Most of these are point mutations within highly conserved regions of the gene that produce altered protein wtth increased stability (4 ), allowing easy detection in affected cells by in immunohistochemical and immnunoblotting techniques. The presence of elevated levels of mutant p53 may itself be a prognostic factor in human breast cancer (5 ,6 ). Furthermore, a significant associatton between high levels of p53 and established pathologrcal crtteria (e.g, tumor stage, estrogen, and progesterone receptor levels) have been described in a number of studies (5 ,6 ). Thus chapter will describe two procedures routinely used in our laboratory for the detection of p53 protein in mammary eptthehal cell lines and in breast-tumor tissue. The methods have been used to analyze p53 levels in breast tumors (5 –7 ) and used in many studies to monitor p53 expression following DNA damage in cell lines (8 –10 ).