Emergence of resistance to antineoplastic drugs poses a major impediment to the successful treatment of breast cancer. We previously reported that human breast carcinoma MCF-7 cells selected for resistance against doxorubicin (MCF-7/DOX cells) expressed high levels of tissue-type transglutaminase (tTGase), a calcium-dependent protein cross-linking enzyme that plays a role in apoptosis. The purpose of this study was to determine the mechanisms by which MCF-7/DOX cells survive and proliferate despite high levels of tTGase expression. Our results demonstrate that the MCF-7/DOX cells contain deficient intracellular calcium pools, which may explain their ability to survive and tolerate the high levels of tTGase expression. Treatment with thapsigargin failed to induce any significant killing of MCF-7/DOX cells. Similar treatment of the drug-sensitive MCF-7 wild-type (MCF-7/WT) cells, however, induced significant apoptosis. Treatment with the ionophore A23187, on the other hand, killed a large percentage of both the MCF-7/DOX and the MCF-7/WT cells. We also established a revertant cell line, MCF-7/RT, from MCF-7/DOX cells to rule out the involvement of P-glycoprotein (P-gp) in these phenomena. Unlike the MCF-7/DOX cells, the MCF-7/RT cells showed no detectable P-gp expression; the MCF-7/RT cells, however, continued to express high levels of tTGase. Moreover, like MCF-7/DOX cells, the MCF-7/RT cells were highly resistant to thapsigargin-induced apoptosis but were sensitive to the ionophore A23187-induced apoptosis. These results suggest that the resistance of MCF7/DOX cells to thapsigargin is linked to their defective intracellular Ca2+ stores, a notion that was directly confirmed by single-cell spectrofluorometric analysis.