PII proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro PII-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of PII signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of PII with its target proteins. The resulting PII-signalling properties indicate that, in mixtures of ADP and ATP, PII trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by PII was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of PII to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on PII–PipX interaction. These results indicate that S. elongatus PII directly senses the adenylate energy charge, resulting in target-dependent differential modification of the PII-signalling properties.