The probability that a cell will undergo apoptosis is in part dictated by the cellular redox potential, which is mainly determined by the reduction and oxidation of thiol residues on glutathione and proteins. We and others have recently shown that mitochondria play a critical role in the apoptotic cascade. Here, we address the question as to whether thiol modification regulates apoptosis and in which cellular compartment apoptosis-regulatory thiols are localized. To resolve this problem, we employed the divalent thiol-reactive agent diamide, which causes thiol cross-linking and thus mimics disulfide bridge formation, and a panel of monovalent thiol-reactive compounds (which impede disulfide bridge formation due to thiol oxidation), one of which is specifically targeted to the mitochondrial matrix. Our data indicate that thymocyte apoptosis induced by diamide mimics natural apoptosis in the sense that mitochondrial transmembrane potential (ΔΨm) disruption precedes nuclear chromatin degradation: that monovalent thiol-reactive compounds inhibit apoptosis induced by diamide, glucocorticoids, irradiation, and topoisomerase inhibition; that the critical thiols determining cell fate after exposure to diamide, glucocorticoids, or DNA damage are likely to be located in the mitochondrial matrix; and that thiol oxidation and reduction are critical for apoptosis induction by some stimuli (glucocorticoids. DNA damage), but not by Fas/CD95 cross-linking. Taken together, these findings suggest that, at least in some pathways of apoptosis, mitochondrial thiols constitute a critical sensor of the cellular redox potential.