The effects of the anti-inflammatory drugs diclofenac, piroxicam, indomethacin, naproxen, nabumetone, nimesulide, and meloxicam on mitochondrial respiration, ATP synthesis, and membrane potential were determined. Except for nabumetone and naproxen, the other drugs stimulated basal and uncoupled respiration, inhibited ATP synthesis, and collapsed membrane potential in mitochondria incubated in the presence of either glutamate + malate or succinate. Plots of membrane potential versus ATP synthesis (or respiration) showed proportional variations in both parameters, induced by different concentrations of nimesulide, meloxicam, piroxicam, or indomethacin, but not by diclofenac. The activity of the adenine nucleotide translocase was blocked by diclofenac and nimesulide; diclofenac also slightly inhibited mitochondrial ATPase activity. Naproxen did not affect any of the mitochondrial parameters measured. Nabumetone inhibited respiration, ATP synthesis, and membrane potential in the presence of glutamate + malate, but not with succinate. NADH oxidation in submitochondrial particles also was inhibited by nabumetone. Nabumetone inhibited O2 uptake in intact cells and in whole heart, whereas the other five drugs stimulated respiration. These observations revealed that in situ mitochondria are an accessible target. Except for diclofenac, a negative inotropic effect on cardiac contractility was induced by the drugs. The data indicated that nimesulide, meloxicam, piroxicam, and indomethacin behaved as mitochondrial uncouplers, whereas nabumetone exerted a specific inhibition of site 1 of the respiratory chain. Diclofenac was an uncoupler too, but it also affected the adenine nucleotide translocase and the H+-ATPase.