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Variations in the rates of atmospheric CH4 uptake in upland soils can arise from both abiotic and biotic factors. Among the less-studied biotic factors is the degree to which methanotroph activity and community composition interact with supply of CH4 to the soil. Here, we investigated whether the abundance of high affinity methanotrophs in a range of soils representing different land use types is substrate (CH4) dependent. Field replicates of three soils sampled from deciduous forest, spruce forest and agricultural sites were incubated in columns flushed continuously for 24 days with air at one of four CH4 concentrations: <1 ppm (starvation), 1.8 (ambient), 30 (low elevated) and 60 (high elevated) ppm. In all soils, CH4 oxidation rates increased linearly with CH4 supply. For all levels of CH4 supply, CH4 oxidation rates were the highest in deciduous forest soil followed by spruce forest and agricultural soils. Terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that the agricultural soil had a distinct methanotrophic community compared to the two forest soils. In particular, the T-RFs (Terminal restriction fragments) associated with USCα and Type II methanotrophs (Methylocystis sp, Methylosinus sp.) were the most abundant in forest soils while Type 1a associated T-RFs dominated in agricultural soil. The agricultural and forest soils also differed in their fractionation of stable isotopes, 13C and 2H, during CH4 oxidation. Altering CH4 concentration in the inlet air did not change methanotroph abundance, as evidenced by three different assays, two qPCR and T-RFLP, that recorded no changes in the number of pmoA genes and/or the relative abundance of T-RFs. Altogether, it is proposed that intrinsic differences in CH4 oxidation rates between soils, particularly between temperate agricultural and forest soils, are driven by methanotroph community structure. The population size of methanotrophs in upland soils did not respond to CH4 availability and is most probably regulated by other factors, such as the availability of nitrogen, cross-feeding or other carbon sources.