A mixed methane-oxidizing biofilm was characterized, concurrently using a number of advanced techniques. Community analysis results by microarray exhibited that type II members dominated the methanotrophic community, in which Methylocystis was most abundant, followed by Methylosinus. Observation results by fluorescent in situ hybridization and confocal microscopy showed multiple biofilm colonies that were irregular, bell-shaped, with mean thickness of approximately 20 mu m. Image analysis results indicated that the relative abundance of methanotrophs peaked at a depth of about 5 mu m. Although the biofilm colonies differed in size, methanotrophs accounted for 4-9%. Gaussian and linear regression results between the biofilm volumes and types I (r (2) = 0.86) and II volumes (r (2) = 0.92), respectively, revealed that type I members played a role in the growth of the biofilm but only below a threshold volume, whereas type II members supported the overall growth. Geostatistical analyses results revealed concentration of types I and II methanotrophic individuals with decreasing depth, and randomness between the spatial locations and population levels. Collectively, the methane-oxidizing biofilm was a highly organized system with methanotrophs and their cohabitants.