Doxorubicin is an anthracycline antibiotic used for cancer chemotherapy. The utility of doxorubicin is limited by its inability to kill all of the cells within a tumor and by resistant cells emerging from the treated population. We have screened for genes that regulate doxorubicin susceptibility in highly tumorigenic breast cancer cells by cDNA microarray and RNA interference (RNAi) analysis, and we have identified genes associated with both proliferation and cell cycle arrest after doxorubicin treatment. We confirmed that MDA-MB-231 cells treated with doxorubicin induce the expression of carbonic anhydrase II (CAII), inhibitor of differentiation/DNA binding 2 (Id2), activating transcription factor 3 (Atf3), and the phosphatidylinositol 3-kinase 55-kDa regulatory subunit p55PIK. These genes were induced at different times and with varying specificities to different chemotherapeutic drugs. In addition to being induced at the transcriptional level, the CAII and clusterin proteins were elevated after doxorubicin treatment. CAII, Id2, p55PIK, and clusterin were not altered by doxorubicin in MCF-7 cells, a weakly tumorigenic cell line used in previous studies of doxorubicin-regulated gene expression. By inhibiting gene expression using RNAi, we found that CAII and clusterin increase cell survival after doxorubicin treatment, whereas Id2 increases susceptibility to doxorubicin. Our results support a model in which highly tumorigenic breast cancer cells induce a transcriptional response to doxorubicin that is distinct from less malignant cells. The induced genes regulate drug susceptibility positively and negatively and may be novel targets for therapeutic intervention.