Mechanisms to protect organisms from the consequences of DNA damage include the tumor suppressor p53 pathway. p53 protein binds specifically to a DNA consensus sequence to induce growth inhibitory genes or nonspecifically to damaged sites leading to DNA repair or apoptosis. While p53 protein is susceptible to post-translational modifications and binding to other proteins, few of the modifications or associations have been demonstrated in the context of the cell. We used a novel, sensitive DNA binding assay to examine p53 proteins in lysates prepared from cells responding to DNA damage. Non-transformed progenitor keratinocytes exhibited rapid and sustained induction of activated p53 protein binding to the consensus sequence, correlated with sustained induction of a downstream target gene, the cyclin-dependent kinase inhibitor p21. Binding to a mismatched DNA probe was transient and correlated with total p53 protein in the lysate, suggesting that most or all of the endogenous p53 proteins induced after damage were capable of mismatched DNA binding. Squamous cell carcinoma (SCC) derivates showed defects in induction p53 protein after DNA damage and disproportional losses in DNA binding, compared to total p53 protein steady state levels, along with increased NDN2/p53 association. Maximum induction of endogenous p53 protein binding to the mismatched probe correlated with transcription-independent induction of apoptosis. We suggest a model in which activated forms of p53 carry out transcription-dependent functions in growth arrest and DNA repair which may vary by cell type, while most or all wild type forms of p53 are capable of binding to damaged DNA. p53 protein loss of binding to damaged DNA causes failure of cells to trigger apoptosis and may lead to resistance to chemotherapy. Implications for new directions in therapeutics include functional molecular profiling of individual patient tumors for p53 DNA binding to predict treatment response better than mutational analysis alone and targeted activation of p53 DNA damage binding in tumor cells for increasing their sensitivity to chemotherapy.