Chronic contractile activity of muscle induces an increase in mitochondria located in proximity to the sarcolemma (SS, subsarcolemmal) and in mitochondria between the myofibrils (IMF, intermyofibrillar). These are energetically favourable metabolic adaptations, but since mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially-mediated apoptotic signaling in muscle. We hypothesized that contractile activity would provide protection against mitochondrially-mediated apoptosis despite an induction of pro-apoptotic proteins. To induce mitochondrial biogenesis, we stimulated (10Hz; 3h/day) rat muscle for 7 days. Contractile activity did not alter the Bax:Bcl-2 ratio, an index of apoptosis, and did not affect manganese superoxide dismutase (MnSOD) levels. However, contractile activity increased anti-apoptotic HSP70 and ARC by 1.3- and 1.4-fold (p<0.05). Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production by 1.4 and 1.9-fold (p<0.05) during state IV and III respiration, respectively, while IMF mitochondrial state IV ROS production was suppressed by 28% (p<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced AIF release (30%) from IMF mitochondria, despite higher inherent cytochrome c and AIF expression. Contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, contractile activity induces predominantly anti-apoptotic adaptations in both mitochondrial subfractions. Our data suggest that chronic contractile activity can exert a protective effect on mitochondrially-mediated apoptosis in muscle.