It is generally accepted that permeabilization of the outer mitochondrial membrane is a crucial event in the induction of apoptosis. In order to know how the cell would respond to the damage targeted not to the outer mitochondrial membrane, but to the mitochondrial interior, we followed the changes in cell morphology and some biochemical parameters triggered by ROS in the mitochondrial inner space. The experiments were carried out in epidermoid carcinoma A431 cells. For stimulated production of singlet oxygen in the inner space of mitochondria, we employed photodynamic treatment (PDT) mediated by a cationic photosensitizer 7-diamino-2,8-dimethyl-5-phenylphenazinium chloride (Safranin O, Safr), accumulating in the inner space of mitochondria. At low to intermediate cytotoxic doses (up to CD50 reducing cellular viability by 50%), Safr-PDT did not reveal hallmarks of dead cells, and the decrease of cellular viability could be attributed to cell cycle inhibition, and enhanced autophagy. High Safr-PDT doses (beyond CD70) did induce cell death by apoptosis involving release of cytochrome c and caspase-3 activation, in addition to enhanced autophagy.