The Synechococcus elongatus mutant (NA3) lacking the nrtABCD gene cluster is defective in active nitrate transport and requires high nitrate concentrations (> 30 mM) for sustained growth. Prolonged incubation of NA3 in a medium containing 2 mM nitrate led to isolation of a pseudorevertant (NA3R) capable of transport of millimolar concentrations of nitrate, from which three mutants with improved affinity for nitrate were obtained. We identified three genes responsible for the latent transport activity for nitrate: ltnA, which encodes a response regulator with no effector domain, ltnB, which encodes a hybrid histidine kinase with two receiver domains, and ltnT, which encodes a sulfate permease-like protein with a putative cyclic nucleoside monophosphate (cNMP)-binding domain. Missense mutations were found in ltnT of the high-affinity derivatives of NA3R, verifying that LtnT acts as the transporter. Overexpression of truncated LtnT lacking the cNMP-binding domain, but not of entire LtnT, conferred nitrate transport activity on NA3, suggesting that the cNMP-binding domain inhibits transport under normal conditions. A nonsense mutation in ltnB, which resulted in elimination of the receiver domains of the encoded protein, was responsible for expression of nitrate transport activity in NA3R. Expression of LtnB derivatives lacking the receiver domains also conferred low-affinity nitrate transport activity on NA3. The phosphoryl group of the histidine kinase domain of LtnB was transferred to Asp52 of LtnA in vitro. Overexpression of LtnA but not of LtnA(D52E) led to manifestation of the latent nitrate transport activity in NA3, indicating involvement of phosphorylated LtnA in activation of the novel transporter.