The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane-matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.