Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeled Prevotella bryantii specific probe PBB14 were used for in situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation of P. bryantii and Butyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system. P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration of P. bryantii cells in vivo (less than 100/nL, i.e. 10(5) per mL).