As energetic metabolism is crucial for muscles, they develop different adaptations to respond to fluctuating demand among muscle types. Whereas quantitative characteristics are known, no study described simultaneously quantitative and qualitative differences among muscle types in terms of substrates utilization patterns. This study thus defined the pattern of substrates preferential utilization by mitochondria from glycolytic gastrocnemius (GAS) and oxidative soleus (SOL) skeletal muscles and from heart left ventrical (LV) in rats. We measured in situ, ADP (2 mM)‐stimulated, mitochondrial respiration rates from skinned fibers in presence of increasing concentrations of pyruvate (Pyr) + malate (Mal), palmitoyl‐carnitine (Palm‐C) + Mal, glutamate (Glut) + Mal, glycerol‐3‐phosphate (G3‐P), lactate (Lact) + Mal. Because the fibers oxygen uptake (V_s) followed Michaelis–Menten kinetics in function of substrates level we determined the V_s and K_m, representing maximal oxidative capacity and the mitochondrial sensibility for each substrate, respectively. V_s were in the order GAS < SOL < LV for Pyr, Glu, and Palm‐C substrates, whereas in the order SOL = LV < GAS with G3‐P. Moreover, the relative capacity to oxidize Palm‐C is extremely higher in LV than in SOL. V_s was not stimulated by the Lact substrate. The K_m was equal for Pyr among muscles, but much lower for G3‐P in GAS and lower for Palm‐C in LV. These results demonstrate qualitative mitochondrial tissue specificity for metabolic pathways. Mitochondria of glycolytic muscle fibers are well adapted to play a central role for maintaining a satisfactory cytosolic redox state in these fibers, whereas mitochondria of LV developed important capacities to use fatty acids. © 2004 Wiley‐Liss, Inc.