We have previously demonstrated that an acute promyelocytic leukemia (APL)-derived cell line NB4 exhibited a relatively higher basal level of reactive oxygen species (ROS) than other leukemia cell lines, which is one of the mechanisms determining a higher apoptotic susceptibility of NB4 cells to arsenic trioxide (ATO)-induced apoptosis. Here we identified the source of the basal ROS generation in NB4 cells. We demonstrated the existence of all the components of phagocytic NADPH oxidase in NB4 cells and found that this oxidase could be effectively activated. The basal ROS generation in NB4 cells could be blocked by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, but not by inhibitors of mitochondria respiratory chain, implying that NADPH oxidase played an essential role in maintaining the basal ROS level in NB4 cells. Furthermore, ATO-induced cytotoxicity was reduced by pre-treatment with DPI in NB4 cells, suggesting the involvement of NADPH oxidase in ATO-induced cytotoxicity. Therefore, increasing the NADPH oxidase activity may be a novel mechanism to enhance cytotoxicity induced by anticancer agents.