This study was undertaken to characterize the consequences of Ral-interacting protein (RLIP76)-loss with respect to drug resistance, transport, radiation resistance, and alternative transport mechanisms in mouse embryonic fibroblasts (MEFs). MEFs were derived from RLIP76+/+, RLIP76+/- and RLIP76-/- mice. The transport of doxorubicin (DOX), colchicine (COL), leukotriene C4 and dinitrophenyl S-glutathione (DNP-SG) was analyzed in inside-out vesicles (IOVs) prepared from MEFs. We used immuno-titration of transport activity to determine the contribution of RLIP76, MRP1, and p-glycoprotein (Pgp) towards total transport activity. Loss of RLIP76 alleles resulted in significant sensitization to radiation, DOX, cisplatin, and vinorelbine (VRL). In IOVs prepared from MEFs, we observed a stepwise loss of transport activity. Loss of RLIP76 confers sensitivity to xenobiotics and radiation due to the loss of a common transport mechanism for glutathione-electrophile conjugates and xenobiotics.