Ammonia switch-off is the immediate inactivation of nitrogen fixation that occurs when a superior nitrogen source is encountered. In certain bacteria switch-off occurs by reversible covalent ADP-ribosylation of the dinitrogenase reductase protein, NifH. Ammonia switch-off occurs in diazotrophic species of the methanogenic Archaea as well. We showed previously that in Methanococcus maripaludis switch-off requires at least one of two novel homologues of glnB, a family of genes whose products play a central role in nitrogen sensing and regulation in bacteria. The novel glnB homologues have recently been named nifI(1) and nifI(2). Here we use in-frame deletions and genetic complementation analysis in M. maripaludis to show that the nifI(1) and nifI(2) genes are both required for switch-off. We could not detect ADP-ribosylation or any other covalent modification of dinitrogenase reductase during switch-off, suggesting that the mechanism differs from the well-studied bacterial system. Furthermore, switch-off did not affect nif gene transcription, nifH mRNA stability, or NifH protein stability. Nitrogenase activity resumed within a short time after ammonia was removed from a switched-off culture, suggesting that whatever the mechanism, it is reversible. We demonstrate the physiological importance of switch-off by showing that it allows growth to accelerate substantially when a diazotrophic culture is switched to ammonia.