In Synechococcus sp. strain PCC 7942, an ATP-binding cassette transporter encoded by the genes nrtA, nrtB, nrtC, and nrtD mediates active transport of nitrate and nitrite, which is inhibited by ammonium, a preferred source of nitrogen for the cyanobacterium. One of the ATP-binding subunits of the transporter, NrtC, has a distinct C-terminal domain of 380 amino acid residues. A mutant NC2, constructed by removal of this domain using genetic engineering techniques, assimilated low concentrations of nitrate and nitrite and accumulated nitrate intracellularly, showing that the domain is not essential for the transporter activities. Assimilation of low concentrations of nitrite was only partially inhibited by ammonium in NC2 but was completely inhibited in the wild-type cells. Cells of NC2 and its derivative (nitrate reductase-less strain NC4) carrying the truncated NrtC but not the cells with the wild-type NrtC accumulated nitrate intracellularly in the presence of ammonium in medium. These findings indicated that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the nitrate/nitrite transporter. In the presence of ammonium, NC2 could not assimilate nitrate despite its ability to accumulate nitrate intracellularly, which suggested that reduction of intracellular nitrate by nitrate reductase is also subject to inhibition by ammonium.