Death receptor CD95 gene expression is frequently low in human breast tumors and is up-regulated by genotoxic treatments in a p53-dependent manner. We have evaluated the relative contribution of promoter and intronic p53 consensus sites to the regulation of the human CD95 gene in breast tumor cells following doxorubicin treatment. Deletion constructs of the promoter region and site-directed mutagenesis of p53 consensus sites in a fragment spanning 1448 bp of the 5'-promoter demonstrate that these sites are not involved in the observed up-regulation of the CD95 gene upon doxorubicin treatment. In contrast, a p53 consensus site located within the first intron of CD95 gene is absolutely required for the inducible expression of CD95 upon genotoxic treatment in breast tumor cells. Analysis of the transcriptional activity of the two most common p53 mutants found in human breast tumors that are associated with resistance to doxorubicin reveals that these mutations completely eliminate the ability of p53 protein to transactivate CD95 gene expression. On the other hand, Bcl-2 overexpression albeit preventing doxorubicin-induced apoptosis, has no effect on p53-mediated CD95 up-regulation in breast tumor cells. Altogether, these results indicate the lack of involvement of p53 consensus sites of the CD95 promoter region and the pivotal role of intronic p53-responsive element in the regulation of human CD95 gene expression in breast tumor cells. Our results also suggest that in breast cancer patients with certain mutations in the p53 gene, expression of death receptor CD95 in response to genotoxic treatments could be severely compromised.