Soil methanotrophic bacteria constitute the only globally relevant biological sink for atmospheric methane (CH4). Nitrogen (N) fertilizers as well as soil moisture regime affect the activity of these organisms, but the mechanisms involved are not well understood to date. In particular, virtually nothing is known about the spatial distribution of soil methanotrophs within soil structure and how this regulates CH4 fluxes at the ecosystem scale. We studied the spatial distribution of CH4 assimilation and its response to a factorial drought × N fertilizer treatment in a 3-year experiment replicated in two grasslands differing in management intensity. Intact soil cores were labelled with 14CH4 and methanotrophic activity mapped at a resolution of ∼100 μm using an autoradiographic technique. Under drought, the main zone of CH4 assimilation shifted down the soil profile. Ammonium nitrate (NH4NO3) and cattle urine reduced CH4 assimilation in the top soil, but only when applied under drought, presumably because NH4+ from fertilizers was not removed by plant uptake and nitrification under these conditions. Ecosystem-level CH4 fluxes measured in the field did show no or only very small inhibitory effects, suggesting that deeper soil layers fully compensated for the reduction in top soil CH4 assimilation. Our results indicate that the ecosystem-level CH4 sink cannot be inferred from measurements of soil samples that do not reflect the spatial organization of soils (e.g. stratification of organisms, processes, and mechanisms). The autoradiographic technique we have developed is suited to study methanotrophic activity in a relevant spatial context and does not rely on the genetic identity of the soil bacterial communities involved, thus ideally complementing DNA-based approaches.