Nitrogen fixation is an essential microbially-mediated process in soils, but we know very little about the microscale distribution of the community involved in this process throughout the soil matrix. It is of particular importance to understand the long-term effect of agricultural disturbance on these communities. A comparative study was set up using three different tillage treatments of an uncultivated grassland, an adjacent plot tilled for the first time and a long-term tilled plot. Soils were sampled at two time points, upon tillage (May 2003) and after the following harvest season (August 2004). They were then separated into four aggregate fractions: macroaggregates larger than 600 µm, two microaggregate fractions (250-600 µm and 75-250 µm) and a clay-silt fraction at <75µm in diameter. Nitrogen-fixing communities were analyzed by constructing clone libraries containing the marker gene for nitrogen fixation, nifH from the sampled microenvironments. In May 2003 a significant community shift was observed in response to tillage between the untilled and newly-tilled plots. Novel sequences of nifH were retrieved from the newly-tilled plot indicating the release of a previously undetected community of nitrogen fixers from the rhizosphere of the previous grass cover. When samples from August 2004 were compared to earlier sampling, little change was observed in the untilled plot. However, the populations in the newly-tilled plot shifted more dramatically from their composition in May 2003. This may be due to a process of stabilization of the diazotrophic communities that is also evidenced by the long-term tilled plot at the initial sampling date.