The nitrogen-fixing, root-associated strain Pseudomonas stutzeri A1501 carries a single gene encoding a protein from the PII family, designated glnK. The glnK gene is co-transcribed with two distantly related copies of amtB genes encoding putative ammonium channels. Transcription of glnK was decreased in the presence of ammonia and was partly dependent on NtrC and RpoN under nitrogen-limiting conditions. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity, auxotrophic for glutamine and unable to deadenylylate glutamine synthetase, while inactivation of amtB1 led to a prototrophic and Nif+ mutant strain. RT-PCR analysis showed that nifA transcription was abolished in the glnK mutant, while glnA remained transcribed. Using the yeast two-hybrid system, an interaction between GlnK and the C-terminal domain of NifL was observed, suggesting GlnK-dependent control of NifA activity by NifL. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen fixation to the glnK mutant, and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in control of ammonia assimilation, of nifA expression and in modulation of NifA activity by NifL.