Freshly isolated mitochondria from guinea pig hearts were assayed as described in the legend of Figure 3 under FET at state 4 and 3 of respiration according to additions indicated by arrows. The experiment was performed either by preincubating the mitochondria with FCCP and triggering ROS production with the substrate, or by successive addition of different concentrations of the uncoupler after energization with substrate. In panel A, the FCCP dose-response curve for mitochondrial ROS is shown for successive additions of the uncoupler (n=4 for each data point, 2 experiments), whereas in panel B, the raw ARed traces obtained after preincubating the mitochondria with the indicated FCCP concentrations is shown. Panels C and D show the behavior of ΔΨm and NAD(P)H, respectively, for the same experiment shown in panel B.