The ability to utilize atmospheric nitrogen (N₂) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N₂ fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ strain SolV was investigated for its ability to fix N₂. Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes, encoding the subunits of the nitrogenase. ‘M. fumariolicum’ SolV was able to fix N₂ at low oxygen (O₂) concentration (0.5 %, v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h⁻¹ (mg cell dry weight)⁻¹]. Based on acetylene reduction assay and growth experiments, the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N₂ fixation within the phylum Verrucomicrobia.