The anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing- bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethane-sulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.