The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14). Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme complex; the remaining subunits make up the peripheral part of the enzyme. We report here on the biochemical characterization and heterologus expression of the Nqo10 subunit. The Nqo10 subunit could not be extracted from the Paracoccus membranes by NaI or alkaline treatment, which is consistent with the presumed membrane localization. By using the maltose-binding protein (MBP) fusion system, the Nqo10 subunit was overexpressed in Escherichia coli. The MBP-fused Nqo10 was expressed in membrane fractions of the host cell and was extractable by Triton X-100. The extracted fusion protein was then isolated by one-step affinity purification through an amylose column. By using immunochemical methods in conjunction with cysteine-scanning mutagenesis and chemical modification techniques, the topology of the Nqo10 subunit expressed in E. coli membranes was determined. The data indicate that the Nqo10 subunit consists of five transmembrane segments with the N- and C-terminal regions facing the periplasmic and cytoplasmic sides of the membrane, respectively. In addition, the data also suggest that the proposed topology of the MBP-fused Nqo10 subunit expressed in E. coli membranes is consistent with that of the Nqo10 subunit in the native Paracoccus membranes. From the experimentally determined topology together with computer prediction programs, a topological model for the Nqo10 subunit is proposed.