Volatile anesthetics may protect the heart against ischemia‑reperfusion injury via the direct action on mitochondrial complexes and by regulating the production of reactive oxygen species (ROS). Recently, we reported that isoflurane induced the attenuation of mitochondrial respiration caused by complex I substrates. This process was not associated with endogenous production of mitochondrial nitric oxide (NO). In the present study, we investigated the effects of isoflurane on mitochondrial respiration and ROS production using complex II substrates. The detailed mechanism of these effects was explored with regards to NO production and the expression of mitochondrial ATP‑dependent K+ (mKATP) channels. Mitochondria were isolated from the heart of Sprague‑Dawley rats. The respiratory rates of mitochondria (0.5 mg/ml) were measured via polarography at 28˚C with computer‑controlled Clark‑type O2 electrodes. The complex II substrate succinate (5 mM) was used; 0.25 mM of isoflurane was administered prior to ADP‑initiated state 3 respiration. The mitochondrial membrane potential (ΔΨm) was measured under treatment with the substrate succinate, or succinate in the presence of the complex I inhibitor rotenone. The detection was achieved in a cuvette‑based spectrophotometer operating at wavelengths of 503 nm (excitation) 527 nm (emission) in the presence of 50 nM of the fluorescent dye rhodamine 123. The H2O2 release rates in the mitochondria were measured spectrophotometrically with succinate, or succinate and rotenone using the fluorescent dye Amplex red (12.5‑25 µM). The results indicated that isoflurane increased the state 3 and 4 respiration rates caused by succinate, which were higher than those noted in the control group in the presence of succinate alone. The NOS inhibitor L‑NIO or the NO‑sensitive guanylyl cyclase 1H‑[1,2,4]oxadiazolo[4,3‑a]quinoxalin‑1‑one did not inhibit the increase in the respiration rate (state 3) induced by isoflurane. The ROS scavengers SPBN and manganese (III) tetrakis (4‑benzoic acid) porphyrin chloride inhibited the increase in the respiration rate (state 3 and 4) induced by isoflurane. This effect was not noted for the putative KATP channel blockers 5‑hydroxydecanoic acid and glibenclamide. Isoflurane caused a greater decrease in the concentration of H2O2 during ADP‑initiated state 3 respiration, and L‑N5‑(1‑Iminoethyl)‑ornithine did not inhibit this effect. In conclusion, isoflurane was determined to modulate mitochondrial respiration and ROS production caused by the complex II substrate succinate. These effects were independent of endogenous mitochondrial NO generation and mitochondrial KATP channel opening.