The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N'-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl-biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3'-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.