The proteasome inhibitor PS-341 inhibits nuclear factor-kappaB (NF-kappaB) activation, induces apoptosis in cancer cells, including multiple myeloma (MM) cells, and has marked clinical activity as a monotherapy for MM. In this study, we found that subtoxic concentrations of PS-341 potently sensitized MM cell lines and patient cells to DNA-damaging chemotherapeutic agents such as doxorubicin and melphalan, including cells resistant to these drugs and those isolated from a patient who had relapsed after PS-341 monotherapy. Moreover, PS-341 abolished cell adhesion-mediated drug resistance. Using gene expression profiling and proteomic analysis, we demonstrate that PS-341, among its other proapoptotic effects, down-regulates the expression of several effectors involved in the cellular response to genotoxic stress. These data suggest that, in addition to down-regulating the expression of apoptosis inhibitors, PS-341 inhibits genotoxic stress response pathways and thereby restores sensitivity to DNA-damaging chemotherapeutic agents. These studies, therefore, provide the framework for clinical use of this agent in combination with conventional chemotherapy.