This biogeochemical, molecular genetic and lipid biomarker study of sediments (similar to 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH4 supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH4 accumulated below. C-14-radiotracer measurements demonstrated active H-2/CO2 and acetate methanogenesis and anaerobic oxidation of CH4 (AOM). Maximum AOM rates occurred near the SMTZ (similar to 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H-2/CO2 and acetate methanogenesis occurred below the SMTZ but H-2/CO2 methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of C-13-depleted CH4 (delta C-13 c.-80 parts per thousand). Areal AOM and methanogenic rates were similar (similar to 1.7 mmol m(-2) day(-1)), hence, CH4 flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to similar to 1.7 m CH4 became progressively more C-13 depleted (delta C-13-82 parts per thousand.) indicating a zone of CH4 recycling which was consistent with the presence of C-13-depleted archaeol (delta C-13-55 parts per thousand). Pore water acetate concentrations decreased in this zone (to similar to 5 mu M), suggesting that H-2, not acetate, was an important CH4 cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.