The objective was to develop a fast and accurate molecular method for the quantification of the intestinal flora in chickens by rRNA fluorescence in situ hybridization (FISH). Seven weeks old conventionally reared Lohmann hens were used to set up the method. To sample ileal intestinal content, the distal part from Meckels diverticulum to the ileo-caecal junction was removed. Fixation was performed in ethanol and phosphate buffered saline. After washing by centrifugation, the sample was resuspended in pre-heated hybridization buffer with oligonucleotide probe labelled with Cy3 (10ng/µl). The cells were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB338, Probe D and Non-EUB338 according to previous publications. Cultures of 38 bacterial strains representing type strains within the genera Bacillus, Listeria, Campylobacter, Bacteroides, Clostridium, Enterococcus, Streptococcus, Escherichia, Salmonella and Lactobacillus were used for optimization of hybridization conditions on slides. Signal was obtained from all 38 reference strains with probe EUB338 and not from the non-sense probe Non-EUB338. In most cases, signal was obtained from the expected target sequence. In the samples from the seven weeks old Lohman hens, 3-7 ×108 bacterial cells per g of sample were counted by the EUB338 probe. Three weeks old male broiler Ross 308 chickens were used to investigate the bacterial composition of the intestine. The birds received a wheat-barley diet. Counts with the EUB338 probe were 1.97x108(std 1.45x108) The means of counts obtained with probes targeting the rRNA of genera Lactobacillus, Bacillus, Enterococcus-Streptococcus, Enterobacteriaceae, Salmonella, Clostridium and Bacteriodes were around 1 ×108 bacterial cells per g of gut material. As for the EUB338 probe, huge variations were observed between the six different samples analysed even though each sample was obtained from a pool of six chickens allocated in the same cage. The technique will be used to investigate the effect of antimicrobials, feed additive, rearing conditions and Salmonella - and Campylobacter status on the bacterial composition of the chicken intestine.