An automated microscopy-based method using fluorescently labelled 16S rRNA-targeted oligonucleotide probes directed against the predominant groups of intestinal bacteria was developed and validated. The method makes use of the Leica 600HR image analysis system, a Kodak MegaPlus camera model 1.4 and a servo-controlled Leica DM/RXA ultra-violet microscope. Software for automated image acquisition and analysis was developed and tested. The performance of the method was validated using a set of four fluorescent oligonucleotide probes: a universal probe for the detection of all bacterial species, one probe specific for Bifidobacterium spp., a digenus-probe specific for Bacteroides spp. and Prevotella spp. and a trigenus-probe specific for Ruminococcus spp., Clostridium spp. and Eubacterium spp. A nucleic acid stain, 4',6-diamidino-2-phenylindole (DAPI), was also included in the validation. In order to quantify the assay-error, one faecal sample was measured 20 times using each separate probe. Thereafter faecal samples of 20 different volunteers were measured following the same procedure in order to quantify the error due to individual-related differences in gut flora composition. It was concluded that the combination of automated microscopy and fluorescent whole-cell hybridisation enables distinction in gut flora-composition between volunteers at a significant level. With this method it is possible to process 48 faecal samples overnight, with coefficients of variation ranging from 0.07 to 0.30.