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Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and β-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans : arginines Arg 87 and Arg 303 in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU + NirC + strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU + NirC − strain. Only the NirC + strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC + strains reduced nitrite up to 10 times more rapidly than isogenic NarU + strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [ 15 N]nitrate and [ 14 N]nitrite revealed that [ 15 N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate–nitrite antiporter, or more likely as a nitrate/H + or nitrite/H + channel.