The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53-mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53-mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of proapoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione conjugate transporter, RLIP76. The RLIP76-p53 complex was showed by both immunoprecipitation analyses of purified proteins and immunofluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα-stimulated transport of glutathione conjugates of 4HNE (GSHNE) and doxorubicin. Drug resistance was significantly greater for p53-mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53-mutant neuroblastoma, based on a model of regulation of p53-induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in overexpression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53-mutant neuroblastoma.