The permanently stratified Lake Kivu is one of the largest freshwater reservoirs of dissolved methane (CH 4 ) on Earth. Yet CH 4 emissions from its surface to the atmosphere have been estimated to be 2 orders of magnitude lower than the CH 4 upward flux to the mixed layer, suggesting that microbial CH 4 oxidation is an important process within the water column. A combination of natural abundance stable carbon isotope analysis (δ 13 C) of several carbon pools and 13 CH 4 -labelling experiments was carried out during the rainy and dry season to quantify (i) the contribution of CH 4 -derived carbon to the biomass, (ii) methanotrophic bacterial production (MBP), and (iii) methanotrophic bacterial growth efficiency (MBGE), defined as the ratio between MBP and gross CH 4 oxidation. We also investigated the distribution and the δ 13 C of specific phospholipid fatty acids (PLFAs), used as biomarkers for aerobic methanotrophs. Maximal MBP rates were measured in the oxycline, suggesting that CH 4 oxidation was mainly driven by oxic processes. Moreover, our data revealed that methanotrophic organisms in the water column oxidized most of the upward flux of CH 4 , and that a significant amount of CH 4 -derived carbon was incorporated into the microbial biomass in the oxycline. The MBGE was variable (2–50%) and negatively related to CH 4 : O 2 molar ratios. Thus, a comparatively smaller fraction of CH 4 -derived carbon was incorporated into the cellular biomass in deeper waters, at the bottom of the oxycline where oxygen was scarce. The aerobic methanotrophic community was clearly dominated by type I methanotrophs and no evidence was found for an active involvement of type II methanotrophs in CH 4 oxidation in Lake Kivu, based on fatty acids analyses. Vertically integrated over the water column, the MBP was equivalent to 16–60% of the average phytoplankton particulate primary production. This relatively high magnitude of MBP, and the substantial contribution of CH 4 -derived carbon to the overall biomass in the oxycline, suggest that methanotrophic bacteria could potentially sustain a significant fraction of the pelagic food web in the deep, meromictic Lake Kivu.