The bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of two domains, a peripheral arm and a membrane arm. NuoH is a counterpart of ND1, which is one of seven mitochondrially encoded hydrophobic subunits, and is considered to be involved in quinone/inhibitor binding. Sequence comparison in a wide range of species showed that NuoH is comprehensively conserved, particularly with charged residues in the cytoplasmic side loops. We have constructed 40 mutants of 27 conserved residues predicted to be in the cytoplasmic side loops of Escherichia coli NuoH by utilizing the chromosomal DNA manipulation technique and investigated roles of these residues. Mutants of Arg(37), Arg(46), Asp(63), Gly(134), Gly(145), Arg(148), Glu(220), and Glu(228) showed low deamino-NADH-K(3)Fe(CN)(6) reductase activity, undetectable NDH-1 in Blue Native gels, low contents of peripheral subunits (especially NuoB and NuoCD) bound to the membranes, and a significant loss of the membrane potential and proton-pumping function coupled to deamino-NADH oxidation. The results indicated that these conserved residues located in the cytoplasmic side loops are essential for the assembly of the peripheral subunits with the membrane arm. Implications for the involvement of NuoH (ND1) in maintaining the structure and function of NDH-1 are discussed.