The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.