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 (approximately 80%) treated with actinomycin D (1 microgram/ml; 48 h) alone in glucose-containing medium, a much smaller percentage of cells (approximately 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 microgram/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.