U937 cells lacking mitochondrial DNA (rho [symbol: see text] cells) are auxotrophic for uridine and pyruvate, hypersensitive to hypoglycemic conditions, and resistant to antimycin A-induced apoptosis. In spite of their obvious metabolic defects, rho [symbol: see text] cells possess a normal mitochondrial transmembrane potential, as well as near-normal capacity to generate superoxide anion after menadione treatment. Similarly to rho + controls, rho [symbol: see text] cells undergo apoptosis in response to tumor necrosis factor-alpha plus cycloheximide. Detailed comparison of the apoptotic process in rho + and rho [symbol: see text] cells reveals essentially the same sequence of events. In response to tumor necrosis factor/cycloheximide, cells first lose their mitochondrial transmembrane potential (delta psi m) and then manifest late apoptotic alterations, such as generation of reactive oxygen species and DNA fragmentation. Experiments involving isolated mitochondria from rho + and rho [symbol: see text] cells confirm that rho [symbol: see text] mitochondria can be induced to undergo permeability transition, a process thought to account for the pre-apoptotic delta psi m disruption in cells. Like rho + mitochondria, rho [symbol: see text] mitochondria contain a pre-formed soluble factor that is capable of inducing chromatin condensation in isolated nuclei in vitro. This factor is released from mitochondria upon induction of permeability transition by calcium or the specific ligand of the adenine nucleotide translocator atractyloside. In conclusion, it appears that all structures involved in the maintenance and pre-apoptotic disruption of the delta psi m, as well as a mitochondrial apoptotic factor(s), are present in rho [symbol: see text] cells and thus are controlled by the nuclear rather than by the mitochondrial genome. These findings underline the contribution of mitochondria to the apoptotic process.