The microbial flora of a fixed-bed anaerobic methanogenic bioreactor fed with acetate/propionate/butyrate was studied by direct, qualitative and quantitative methods avoiding culture isolation. The aims were to identify species, determine the distribution of microbes between the biofilm attached to the substratum and the free flocs, and define the acidogenic, acetogenic and methanogenic contingents. Optical and scanning electron microscopies showed heterogeneous assemblies of microbes in the biofilm and flocs, which were confirmed by antigenic fingerprinting. A diversity of species involved in the three phases of methanogenesis was detected, and most of these species were antigenically different from the reference organisms. Some microbial subpopulations identified by antigenic fingerprinting changed in size within an interval of 3 weeks, i.e. they either increased or decreased their concentrations by at least tenfold, while others remained relatively constant. The total cell concentration in the flocs was lower than in the biofilm, but at least one microbial subpopulation was more concentrated in the former than in the latter, indicating a preference of location between the compartments available within the bioreactor.