Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O₂]. In solutions containing glucose and cells, [O₂] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2−4 h of incubation with 20 μM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation.