Alterations in intracellular Ca2+ homeostasis occur in a variety of malignancies, including prostate cancer. Although the specific nature of Ca2+ regulation in malignant cells appears to be complex, a common underlying theme is a general loss in their requirement of extracellular Ca2+ for cell growth control. Use of thapsigargin (TG), a potent and specific inhibitor of intracellular Ca2+ ATPases, i.e. sarcoplasmic/endoplasmic reticulum Ca 2+ ATPases (SERCA), which play a central role in modulating cellular Ca2+ homeostasis, has allowed analysis of various SERCA-dependent cellular functions. In our first study, we demonstrate that TG-mediated endoplasmic reticulum (ER) Ca2+ pool depletion induces activation of death receptor 5 (DR5) and mitochondrial pathways; upregulation of DR5 occurs through increasing DR5 mRNA stability and induction of the DR5 genomic promoter-reporter gene, in human cancer cells. Although TG can induce apoptosis, we have developed in a stepwise manner human prostate cancer cell lines (PC3/TG and DU145/TG) that are highly resistant to TG, and revertants of PC3/TG2000 (designated PC3/TGRev) and DU145/TG (designated DU145/TG Rev) cells by growing them in culture continuously in the absence of TG to better understand the TG-mediated cellular adaptive responses. This study reports that TG resistance in the PC3 model is a reversible phenotype with SERCA overexpression contributing only partly to the overall resistance, while DU145/TG cells show an irreversible TG resistant phenotype. Even though PC3 and DU145 cells are both of prostate origin and androgen-independent, they demonstrate diverse set of responses with respect to gene expression profiles during TG selection; some of these changes may be directly involved in mediating TG-resistance, while other changes in gene expression likely represent an epiphenomena of the selection process. The present studies demonstrate the cellular consequences of SERCA inhibition as they relate to apoptosis and resistance to apoptosis increasing our understanding of the role of intracellular Ca2+ homeostasis in such biological processes in cancer cells.