In the present report, we demonstrate that reduction of cellular ATP content with antimycin A blocks actinomycin D-induced apoptotic cell death in HeLa cells. Compared to cells (˜80%) treated with actinomycin D (1 μg/ml, 48 h) alone in glucose-containing medium, a much smaller percentage of cells (˜20%) treated with actinomycin D in the presence of antimycin A in glucose-free medium shows morphological characteristic of apoptosis. ATP-depleted cells with or without actinomycin D treatment, on the other hand, die necrotically. In cells under actinomycin D short exposure treatment (1 μg/ml; 1 h), apoptosis occurs when cellular ATP content is rapidly recovered after the removal of antimycin A and resupplementation of glucose-containing medium. In the incubation of isolated Triton-permeabilized cells with ATP (>0.5 mM), apoptotic nuclei become abundant 4 h after ATP treatment. These results implicate the requirement of ATP for the induction of apoptosis.