We have observed rapid and extensive depletion of cellular energy stores by ⁹-tetrahydrocannabinol (THC) in the pulmonary transformed cell line A549. ATP levels declined dose dependently with an IC₅₀ of 7.5 µg/ml of THC after 24-h exposure. Cell death was observed only at concentrations >10 µg/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 µg/ml. At concentrations of 2.5 or 10 µg/ml of THC, a decrease in mitochondrial membrane potential was observed as early as 1 h after THC exposure. Mitochondrial function remained diminished for at least 30 h after THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicate that JC-1 red fluorescence was fivefold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract. Comparison with a variety of mitochondrial inhibitors demonstrates that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 h after removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences.