Tetrazolium reduction assays, phospholipid analysis, and 16S rRNA (rDNA) sequence analysis were applied to assess the distribution, composition and activity of microbial communities developing in biofilters treating non-ozonated and ozonated drinking water, The response of media-attached biomass to both operating temperature (3 degrees C vs. > 12 degrees C) and ozone application point was assessed. As judged by 2-(p-iodo-phenyl)-3-(p-nitrophenyl)-s-phenyl tetrazolium chloride (INT) reduction, the dehydrogenase activity in biofilter systems that were operated with non-ozonated water was 55% lower than in identical filters operating with ozonated water. There was no significant difference between the microbiological activity measured in a biofilter series treating ozonated water and an identical series where ozonated water was introduced at an intermediate point. The biomass levels in biofilter systems that were operated with ozonated water were 47% higher on average than identical systems operated with non-ozonated water. Operating temperature had no significant impact on total biomass levels however, specific dehydrogenase activity was 70% higher in systems operated at ambient temperatures ( > 12 degreesC) than in systems held at 3 degrees C. Phospholipid and rDNA analysis suggests that there was a community structure response to ozone application and operating temperature, but no response to different ozone application points. (C) 2001 Elsevier Science Ltd. All rights reserved.