The mesophilic methanogenic archaeonMethanosarcina mazeistrain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster inM. mazeiGö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities tonifH, nifI1,nifI2,nifD,nifK,nifEandnifN, similar to other diazotrophic methanogens and certain bacteria such asClostridium acetobutylicum, with the twoglnB-like genes (nifI1andnifI2) located betweennifHandnifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes ofM. mazeiGö1 showed that they are most closely related toMethanosarcina barkeri nif2genes, and also closely resemble those for the correspondingnifproducts of the gram-positive bacteriumC. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that theM. mazei nifgenes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of theM. mazei nifHgene, which may have a function in transcriptional regulation of thenifoperon.