We measured d 13 C of CO 2 , CH 4 , and acetate-methyl in profundal sediment of eutrophic Lake Dagow by incubation experiments in the presence and absence of methanogenic inhibitors chloroform, bromoethane sulfonate (BES), and methyl fluoride, which have different specificities. Methyl fluoride predominantly inhibits acetoclastic methanogenesis and affects hydrogenotrophic methanogenesis relatively little. Optimization of methyl fluoride concentrations resulted in complete inhibition of acetoclastic methanogenesis. Methane was then exclusively produced by hydrogenotrophic methanogenesis and thus allowed determination of the fractionation factors specific for this methanogenic pathway. Acetate, which was then no longer consumed, accumulated and allowed determination of the isotopic signatures of the fermentatively produced acetate. BES and chloroform also inhibited CH 4 production and resulted in accumulation of acetate. The fractionation factor for hydrogenotrophic methanogenesis exhibited variability, e.g., it changed with sediment depth. The d 13 C of the methyl group of the accumulated acetate was similar to the d 13 C of sedimentary organic carbon, while that of the carboxyl group was by about 12% higher. However, the d 13 C of the acetate was by about 5% lower in samples with uninhibited compared with inhibited acetoclastic methanogenesis, indicating unusual isotopic fractionation. The isotope data were used for calculation of the relative contribution of hydrogenotrophic vs. acetoclastic methanogenesis to total CH 4 production. Contribution of hydrogenotrophic methanogenesis increased with sediment depth from about 35% to 60%, indicating that organic matter was only partially oxidized in deeper sediment layers.Eutrophic lakes usually develop an anoxic hypolimnion that lasts over much of the summer season. During the rest of the year, when the hypolimnion is oxic, only the top few centimeters of the sediment become oxidized while the rest remains reduced. In these sediments, production of CH 4 is the terminal step in the degradation of organic matter, and carbon cycling in the lakes is greatly affected by the release of CH 4 from the sediment into the water column (Rudd and Taylor 1980). Organic matter is fermented to acetate, H 2 , and CO 2 , which are subsequently converted to CH 4 . The relative contribution of acetate and H 2 plus CO 2 to CH 4 production can vary, and a mechanistic explanation for this behavior is not always available (Conrad 1999). The relative contribution of either acetoclastic or hydrogenotrophic methanogenesis also affects the d 13 C of the produced CH 4 (Whiticar 1999;Hornibrook et al. 2000). During hydrogenotrophic methanogenesis (4H 2 + CO 2 R CH 4 + 2H 2 O) the isotopically lighter carbon is strongly preferred, whereas the isotope effect is less expressed in acetoclastic methanogenesis (CH 3 COOH R CH 4 + CO 2 ), in which CH 4 is almost exclusively produced from the methyl group of the acetate (Weimer and Zeikus 1978;DeGraaf et al. 1996). Hence, the wide range of differ...