Aerated organic biofilters treating pig manure exhibit partial nitrogen removal. In order to optimize this process, a better comprehension of its colonization by denitrifiers was needed. Three pilot aerated biofilters, fed with variable Biological Oxygen Demand after five days: Total Kjeldahl Nitrogen (BOD5:TKN) ratios, were constructed and monitored during 180 days. Nitrogen was analyzed in the gaseous and liquid flows, at different depths in the systems. Denitrifying biomass was characterized by evaluating its observed (nitrogen mass balances) and potential (adapted acetylene inhibition technique) activities and its quantity (real-time PCR on nirS), at different heights inside the biofilters. Denitrification was observed as soon as nitrate was produced by nitrifiers, after approximately 40 days of operation, but the potential to denitrify increased from the beginning of the monitoring period. Biofilter fed with the highest BOD5:TKN ratio showed significant differences with the others, particularly after 80 days of operation, as its potential activity was lower with a higher observed nitrate removal. Data showed that denitrifiers were mainly localized near the surface of the filter and that a microbiological gradient was present from top to bottom. The potential denitrifying activities were always higher than what was being observed inside the sections studied, suggesting that the biomass could have reduced more nitrate and that conditions found inside the filter did not allow denitrification to completely occur. (c) 2007 Elsevier Ltd. All rights reserved.