In the cyanobacterium Synechocystis sp. strain PCC 6803 we have previously reported the presence of two different proteins with glutamine synthetase activity: GSI, encoded by the glnA gene, and GSIII, encoded by the glnN gene. In this work we show that expression of both the glnA and glnN genes is subjected to transcriptional regulation in response to changes in nitrogen availability. Northern blot experiments and transcriptional fusions demonstrated that the glnA gene is highly transcribed in nitrate- or ammonium-grown cells and exhibits two- to fourfold-higher expression in nitrogen-starved cells. In contrast, the glnN gene is highly expressed only under nitrogen deficiency. Half-lives of both mRNAs, calculated after addition of rifampin or ammonium to nitrogen-starved cells, were not significantly different (2.5 or 3.4 min, respectively, for glnA mRNA; 1.9 or 1.4 min, respectively, for glnN mRNA), suggesting that changes in transcript stability are not involved in the regulation of the expression of both genes. Deletions of the glnA and glnN upstream regions were used to delimit the promoter and the regulatory sequences of both genes. Primer extension analysis showed that structure of the glnA gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified, Escherichia coli-expressed Synechocystis NtcA protein binds to the promoter of the glnA gene. Primer extension also revealed the existence of a sequence related to the NtcA binding site upstream from the glnN promoter. However, E. coli-expressed NtcA failed to bind to this site. These findings suggest that an additional modification of NtcA or an additional factor is required for the regulation of glnN gene expression.