This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (SOL) and superficial white gastrocnemius (WG). Threshold for Ca²⁺-induced mitochondrial Ca²⁺ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were ~3 fold higher in fibers from WG compared to SOL (124 ± 47 vs. 30.4 ± 6.8 pmol Ca²⁺/mU citrate synthase (CS)). A similar phenomenon was also observed in isolated mitochondria (threshold 121 ± 60 vs. 40 ± 10 nmol Ca²⁺/mg prot. in WG and SOL), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative proteins modulators (CypD, ANT-1 and VDACs) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the SOL. However, endogenous matrix Ca²⁺ measured in mitochondria isolated under resting baseline conditions was ~2-fold lower in the WG than in the SOL (56 ± 4 vs.111 ± 11 nmol/mg prot), which significantly accounted for the resistance of WG. Taken together these results reveal fiber type differences in the sensitivity to Ca²⁺-induced-PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca²⁺ dynamics between fiber types.