The status of the Archaea as one of the three primary Domains emphasizes the importance of understanding their molecular fundamentals. Basic transcription in the Archaea resembles eucaryal transcription. However, little is known about transcriptional regulation. We have taken an in vivo approach, using genetics to address transcriptional regulation in the methanogenic Archaeon Methanococcus maripaludis. We identified a repressor binding site that regulates nif (nitrogen fixation) gene expression. The repressor binding site was palindromic (an inverted repeat) and was located just after the transcription start site of nifH. Mutations that changed the sequence of the palindrome resulted in marked decreases in repression by ammonia, even when the palindromic nature of the site was retained. The same mutations greatly decreased binding to the site by components of cell extract. These results provide the first partial description of a transcriptional regulatory mechanism in the methanogenic Archaea. This work also illustrates the utility of genetic approaches in Methanococcus that have not been widely used in the methanogens: directed mutagenesis and reporter gene fusions with lacZ.