The last two decades have led to a greater understanding of the genetic basis of human malignancy. Although numerous genetic alterations have been detected in cancer, activation of oncogenes and inactivation of cell cycle regulators (e.g., tumor suppressor genes) are now known to play a critical role in the progression of the disease. Therapeutic strategies based on specific molecular alterations in cancer include reintroduction of wild-type tumor suppressor function to cells lacking the gene. p53 gene therapy provides an attractive strategy to test the potential clinical feasibility of this approach. Alterations in p53 function are present in approximately half of all malignancies, and expression of wild-type p53 can result in apoptosis in human tumor cells. This review summarizes current investigations with p53 gene therapy, highlighting the preclinical efforts with adenoviral, retroviral, and lipid-based gene delivery systems. A comprehensive review of the various clinical targets suggested for p53 gene therapy is presented together with challenges and prospects for future clinical investigation.