We have shown previously that down-regulation of CK2 activity (protein kinase CK2, formerly casein kinase 2) by employing its inhibitors apigenin or 4,5,6, 7-tetrabromobenzotriazole promotes apoptosis in prostatic carcinoma cells. In an effort to define the downstream mediators of this action, we show that cell apoptosis observed on down-regulation of CK2 is preceded by intracellular generation of hydrogen hydroxide (H2O2) in various normal and cancer cells. In this regard, both androgen-dependent ALVA-41 and androgen-independent PC-3 cells treated with 80 μmol/L apigenin or 4,5,6,7-tetrabromobenzotriazole or with antisense CK2α oligonucleotide or small interfering RNA respond similarly to down-regulation of CK2. Interestingly, whereas chemical inhibitors of CK2 elicited H2O 2 production in both cancer and noncancer cells, the antisense CK2α-mediated down-regulation of CK2 showed significant H 2O2 production in cancer cells but had minimal effect in noncancer cells. The basis of this key difference is unclear at present, but this observation may have implications for the therapeutic potential of antisense CK2 oligonucleotide in cancer therapy. The H2O2 production induced by antisense CK2α was associated with robust caspase-3 activity, nuclear factor-κB nuclear translocation, cytochrome c release, and subsequent DNA fragmentation in prostate cancer cells (ALVA-41 and PC-3). These findings describe, for the first time, a relationship between CK2 and reactive oxygen species, such that CK2 inhibition leads to production of intracellular H2O2, which may serve as a downstream mediator of apoptosis in cancer cells.