Despite its central role in biotiltration, the ecology of the microbial community in biofilters remains largely unknown, primarily because of the difficulty of making detailed observations. Recently developed molecular techniques now allow determination of community composition from DNA extracted from the biofilter, without the need to culture the organisms. This study examined the structure of the microbial communities in ethanol biofilters through the application of the molecular fingerprinting technique, automated ribosomal RNA intergenic spacer analysis (ARISA). Three communities were tracked through acclimation to ethanol in liquid culture, and two of these were followed through growth within two biotilters packed with sand or lava rock. One of these created a successful set of biofilters while the other failed because of acidification. Analysis of the fingerprints revealed a strong decrease in species diversity towards the end of the acclimation period. Diversity indices show a rise in diversity for the biofilter with inconsistent removal of ethanol and low diversities in the successful biofilters, which removed ethanol with an efficiency near 80% for both packing materials. The results indicate that community fingerprinting shows promise as a means of assessing biofilter microbial communities. (c) 2005 Elsevier B.V. All rights reserved.