AimsTo develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. Methods and ResultsNew FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. ConclusionsSimultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. Significance and Impact of the StudyThis is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.