The metabolite succinate accumulates during tissue ischemia. In early reperfusion, succinate is rapidly metabolized, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. It is widely believed that the source of succinate-driven ROS in early reperfusion is reverse electron transfer (RET) at respiratory complex I (Cx-I). However, evidence for Cx-I RET ROS is largely based on experimental evidence from isolated mitochondria with succinate as the sole substrate. We hypothesized that numerous other conditions present during early reperfusion may impact Cx-I RET ROS. Thus, experimental perturbations were applied to mitochondria to model early reperfusion, with measurement of ROS via the fluorescent probe PHPA. These conditions included: (i) A high concentration of NADH; (ii) A high concentration of lactate; (iii) A mildly acidic pH; (iv) A non-zero concentration of ADP buffered by a creatine / creatine phosphate / creatine kinase system; (v) Presence of purine nucleotide breakdown products; (vi) Presence of calcium. These studies revealed that under early reperfusion-like conditions mitochondrial ROS generation still occurred via Cx-I RET, but at a magnitude only half that previously reported. Furthermore, residual ROS that was insensitive to a specific Cx-I RET inhibitor (S1QEL) was slightly elevated under early-reperfusion like conditions, and this residual ROS was mostly inhibited by S3QEL, a blocker of ROS from respiratory complex III (Cx-III). Together, these data suggest that the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and that of Cx-III ROS underestimated.