Pure singlet molecular oxygen (1O2) generated by thermal decomposition of the 3,3'-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inhibited respiration of isolated rat liver mitochondria supported by NADH-linked substrates or succinate, but not by N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbate. Under the latter conditions, mitochondria treated with 2.7 mM NDPO2 exhibited a decrease in transmembrane potential (deltapsi) in manner dependent on NDPO2 exposure time. This process was sensitive to the mitochondrial permeability transition inhibitors EGTA, dithiothreitol, ADP, and cyclosporin A. The presence of deuterium oxide (D2O), that increases 1O2 lifetime, significantly enhanced NDPO2-promoted mitochondrial pereabilization. In addition, NDPO2-induced mitochondrial permeabilization was accompanied by DTT or ADP-sensitive membrane protein thiol oxidation. Taken together, these results provide evidence that mitochondrial permeability transition induced by chemically generated singlet oxygen is mediated by the oxidation of membrane protein thiols.