Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40-50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by approximately 50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.