The inhibitory sites of component D1, isolated by extracting burn eschar with ethyl acetate, on rat liver mitochondrial respiratory chain was investigated. By using changes of the redox state of NADPH in various respiratory states, the effect of D1 on electron flow through the phosphorylation site 1 was studied. It was found that D1 did not interfere with electron transport through site 1, nor did it inhibit the activity of NADH:duroquinone reductase. The functional state of site 2 was tested directly by using duroquinol as an artificial electron donor and cytochrome c as electron acceptor. D1 inhibited the activity of this enzyme in a dose-dependent manner. The redox state of the electron carrier cytochrome b was not influenced by D1. However, a transient process of oxidation of cytochrome c1 was demonstrated, suggesting a brief inhibitory process in the reducing side of it. Two inhibitory sites in site 2 were found by studying the TMPD and DCIP bypasses. One was at the reducing side of cytochrome c1, and the other was at the reducing side of cytochrome b. Further studies are needed to find out the exact position and nature of these inhibitory sites.