The purpose of this study was to determine if regular exercise (treadmill running, 10 weeks) alters the susceptibility of rat isolated heart mitochondria to Ca²⁺-induced PTP opening and if this could be associated with changes in the modulation of PTP opening by selected physiological effectors. Basal leak-driven and ADP-stimulated respiration in the presence of substrates for complex I, II and IV were not affected by training. Fluorimetric studies revealed that in the control (C) and exercise-trained (T) groups, the amount of Ca²⁺ required to trigger PTP opening was greater in the presence of complex II vs I substrates (230 ± 12 vs 134 ± 7 nmol Ca²⁺.mg prot.-1 respectively, P < 0.01, pooled average of C and T groups). In addition, with a substrate feeding the complex II, training increased by 45 % (P < 0.01) the amount of Ca²⁺ required to trigger PTP opening, both in the presence and absence of the PTP inhibitor cyclosporin A. However, , ROS production, NAD(P)H ratio and Ca²⁺ uptake kinetics were not different in mitochondria from both groups. Taken together, these results suggest the existence of a substrate-specific regulation of the PTP in heart mitochondria and that regular exercise results in a reduced sensitivity to Ca²⁺-induced PTP opening in presence of complex II substrates.