The permeabilized cells and muscle fibers technique allows to study the functional properties of mitochondria without their isolation, thus preserving intact all contacts with cellular structures, mostly cytoskeleton, to study the whole population of mitochondria in the cell in their natural surrounding and is increasingly used both in experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibers are very different from those in isolated mitochondria in vitro. In this article we analyze the data showing the dependence of this parameter upon muscle contractile state. Most remarkable is the effect of recently described calcium- independent contraction of permeabilized muscle fibers induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous calcium-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibers that resulting in a disorganization of mitochondrial arrangement leading to significant increase of affinity for ADP. These data show that calcium-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore, use of their inhibitors is strongly advised at the step of permeabilization with careful washing of fibers or cells afterwards. Possible physiologically relevant relationship between calcium-regulated, ATP-dependent contraction and mitochondrial functional parameters is also discussed.