Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76-/- MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis - RLIP76-/- mice will be deficient in chemical neoplasia - was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76-/-. PMA-induced skin carcinogenesis in RLIP76+/+ mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76-/- mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76-/- mice. In RLIP76-/- mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.