The deleterious action of Cd2+ on rat liver mitochondria was investigated in this work using spectroscopic and microscopic methods. The concentration dependence of Cd2+ on mitochondrial swelling, membrane potential and membrane fluidity was studied. Our aim was to detect the active sites of Cd2+ in the mitochondrial membrane treatments with cyclosporin A (CsA) and EGTA on the mitochondrial permeability transition (MPT) induced by low and high concentrations of Cd2+. The protective effects of dithiothreitol, human serum albumin and monobromobimane+ on Cd2+-induced MPT were also monitored. All of these investigations indicated that Cd2+ can directly affect MPT at two separate localization sites at different concentrations: the classic Ca2+ triggering site and the thiol (-SH) groups of membrane proteins matched by MPT pore opening (defined as "S" site). At the high concentration of Cd2+, other free -SH groups in the mitochondrial matrix may be involved in this process. These findings were supported by transmission electron microscopy and shed light on the toxic mechanism of Cd2+ on mitochondria.