Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to P(II) protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.