Forest ecosystems assimilate more CO 2 from the atmosphere and store more carbon in woody biomass than most nonforest ecosystems, indicating strong potential for afforestation to serve as a carbon management tool. However, converting grasslands to forests could affect ecosystem-atmosphere exchanges of other greenhouse gases, such as nitrous oxide and methane (CH 4 ), effects that are rarely considered. Here, we show that afforestation on a well-aerated grassland in Siberia reduces soil CH 4 uptake by a factor of 3 after 35 years of tree growth. The decline in CH 4 oxidation was observed both in the field and in laboratory incubation studies under controlled environmental conditions, suggesting that not only physical but also biological factors are responsible for the observed effect. Using incubation experiments with 13 CH 4 and tracking 13 C incorporation into bacterial phospholipid fatty acid (PLFA), we found that, at low CH 4 concentrations, most of the 13 C was incorporated into only two PLFAs, 18 : 1x7 and 16 : 0. High CH 4 concentration increased total 13 C incorporation and the number of PLFA peaks that became labeled, suggesting that the microbial assemblage oxidizing CH 4 shifts with ambient CH 4 concentration. Forests and grasslands exhibited similar labeling profiles for the high-affinity methanotrophs, suggesting that largely the same general groups of methanotrophs were active in both ecosystems. Both PLFA concentration and labeling patterns indicate a threefold decline in the biomass of active methanotrophs due to afforestation, but little change in the methanotroph community. Because the grassland consumed CH 4 at a rate five times higher than forest soils under laboratory conditions, we concluded that not only biomass but also cell-specific activity was higher in grassland than in afforested plots. While the decline in biomass of active methanotrophs can be explained by site preparation (plowing), inorganic N (especially NH 4 1 ) could be responsible for the change in cell-specific activity. Overall, the negative effect of afforestation of upland grassland on soil CH 4 uptake can be largely explained by the reduction in biomass and to a lesser extent by reduced cell-specific activity of CH 4 -oxidizing bacteria.