Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular Ca2⁢ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular Ca2⁢ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a Ca2⁢ activated protein, and Ca2⁢/ calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of Ca2⁢/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular Ca2⁢ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and Ca2⁢/calmodulin-dependent protein kinase II in HepG2 cells.