Azotobacter vinelandii has three nitrogenases: a molybdenum (Mo) nitrogenase, a vanadium (V) nitrogenase, and a third nitrogenase (nitrogenase-3), which apparently lacks Mo and V. Mo represses synthesis of both V nitrogenase and nitrogenase-3, and in the absence of Mo, V represses synthesis of nitrogenase-3. We have investigated transcriptional regulation of the three nitrogenases by metals using Northern analysis and probes specific for transcripts of each of the three nitrogenases. Our results confirm that Mo is required for expression of the Mo nitrogenase structural genes (nifHDK), and substantiate the notion that Mo represses transcription of the structural genes for both V nitrogenase and nitrogenase-3. We show that repression by V of nitrogenase-3 is also effected at the level of transcription. Unexpectedly, V only represses transcription of the nitrogenase-3 structural genes (anfHDGK) if the V nitrogenase structural gene cluster vnfDGK is present. Further, deletion of nifHDK allows low expression of anfHDGK in the presence of Mo. Repression by Mo or V is independent of cofactor synthesis and therefore of enzyme activity.