The cyanobacterial protein NtcA is a global transcriptional regulator of genes involved in nitrogen assimilation that are subjected to ammonium-promoted repression and is itself controlled by positive autoregulation. Strain CSI70 derived from Synechococcus sp. strain PCC 7942 was constructed to overexpress an additional ntcA gene copy from a constitutive promoter. This strain exhibited high levels of the NtcA protein both in the presence and in the absence of ammonium. However, expression of the NtcA-dependent nir operon and glnA gene (tested by RNA/DNA hybridization and enzyme activity) was still subjected to nitrogen regulation. These results indicate in vivo regulation of the activity of NtcA at activation of transcription of nitrogen-regulated genes as a function of the nitrogen status of the cell.