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The impacts of land-use change and long-term agricultural practices on the bacterial and methanotrophic biomass in the soils comprising the Classical Experiments at Rothamsted Research were investigated by using 13CH 4 phospholipid fatty acid stable isotope probing (PLFA SIP). Nine sites were studied, including six arable plots, two regenerated woodlands and a regenerated grassland. The regenerating sites had all been in arable cropping for at least 200 years (and probably much longer) before commencing regeneration approximately 120 years ago. Six sites were plots within the Broadbalk Wheat Experiment, which had been cultivated, and on which winter wheat was grown annually for 163 years with different fertilizer and manure treatments; the field had been in arable cropping for at least 200 years previously. Triplicate soil samples were incubated under 2 ppmv 13CH 4 for up to 100 days. Extraction and 13C-PLFA analysis revealed that overall methanotrophic biomass was smaller in the Rothamsted soils compared with the other mineral soils studied to date, as indicated by poor 13C incorporation. High-affinity methanotrophs similar to known type II methanotrophs were most abundant in all of the soils studied, except in two plots receiving farmyard manure (FYM and FYM + N). Manuring resulted in a shift to a population similar to known type I methanotrophs. Methanotrophic biomass was elevated in soils that had received the largest input of N fertilizer, though without detectable differences in CH 4 oxidation rates, indicating the potential mediation of atmospheric CH 4 oxidation by non-methanotrophs, which are most likely to be nitrifying bacteria. © 2011 The Authors. Journal compilation © 2011 British Society of Soil Science.