The NDI1 gene encodes the internal rotenone-insensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADH-ubiquinone-1 (UQ1) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH-UQ1 reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. coli membranes are different from S. cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition.