Abstract. Lake sediments in Amazonia are a significant source of CH4, a potential greenhouse gas. Previous studies of sediments using 13C analysis found that the contribution of hydrogenotrophic versus aceticlastic methanogenesis to CH4 production was relatively high. Here, we determined the methanogenic pathway in the same sediments (n = 6) by applying [14C]bicarbonate or [2-14C]acetate, and confirmed the high relative contribution (50–80 %) of hydrogenotrophic methanogenesis. The respiratory index (RI) of [2-14C]acetate, which is 14CO2 relative to 14CH4 + 14CO2, divided the sediments into two categories, i.e., those with an RI 0.4 showing that a large percentage of the acetate-methyl was oxidized to CO2 rather than reduced to CH4. Hence, part of the acetate was probably converted to CO2 plus H2 via syntrophic oxidation, thus enhancing hydrogenotrophic methanogenesis. This happened despite the presence of potentially aceticlastic Methanosaetaceae in all the sediments. Alternatively, acetate may have been oxidized with a constituent of the sediment organic matter (humic acid) serving as oxidant. Indeed, apparent acetate turnover rates were larger than CH4 production rates except in those sediments with a R