Changes to microbial populations in a two-phase anaerobic digestion system were studied over 34 weeks. Numbers of autofluorescent methanogenic and non-methanogenic bacteria decreased significantly during start-up, but did not change markedly either in the acid reactor or the upflow anaerobic filter for the remainder of the study. Although the proportion of autofluorescent methanogens increased in the acid reactor, the numbers of viable methanogens decreased 590-fold. The numbers of viable methanogens increased 10-fold in the port, decreased 10-fold in the effluent and there was almost no change in the drain of the upflow anaerobic filter. The data indicated that bacterial attachment in the upflow anaerobic filter gave a 90% COD removal and a methane yield of 0.33 m(3) CH(4)kg(-1) COD removed at an organic loading rate of 7 kg COD m(-3) day(-1). Epifluorescence microscopy of the seed sludge revealed a diverse methanogenic population of equally dominant groups of medium rods and filaments with Methanococcus, short rods, Long rods and Methanosarcina also present. The medium rod-shaped species remained the most dominant group in the acid reactor. As the volatile fatty acid concentration increased in the acid reactor the number of Methanosarcina and filament species decreased, becoming the least dominant groups. At the end of the operation, Methanococcus species were the dominant group in the upflow anaerobic filter having been washed from the biofilm. (C) 2000 Society of Chemical Industry.