Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methaneconsuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56% ؎ 8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94% ؎ 2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.Large amounts of methane are stored in the subsurface ocean as crystalline gas hydrate, dissolved in porewater, and as free gas. Most of the methane which rises from subsurface reservoirs is consumed by anaerobic microorganisms inhabiting sulfate-penetrated sediment layers before it reaches the seafloor (65). This microbially mediated anaerobic oxidation of methane (AOM) controls the emission of the greenhouse gas methane from the ocean to the atmosphere (see reference 23 and references therein).In marine habitats, the metabolic process of AOM is proposed to be a reversed methanogenesis coupled to the reduction of sulfate involving methanotrophic archaea (ANME archaea) and sulfate-reducing bacteria (SRB). ANME archaea and SRB are assumed to interact syntrophically (26) and form microbial consortia which oxidize methane with equimolar amounts of sulfate, yielding bicarbonate and sulfide (26, 51, 52). Sulfide produced as a by-product of AOM at cold seeps often supports chemosynthetic communities which derive energy from sulfide oxidation. ...