The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro. 2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate. 3. Mitochondrial respiration inhibition was dependent upon alteration of -SH groups. 4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane. 5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.