In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA. Certain mutations in nifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of nif gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination of nifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifH mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH₄⁺ uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.