bThe development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. Ciliate protozoa have been found to colonize the intestinal tracts of a wide range of ruminant and nonruminant herbivores (1). In the rumen ecosystem, ciliates can account for up to 50% of the total microbial nitrogen, reaching densities of 10 5 to 10 6 cells/ml rumen fluid (2, 3). Although ciliates are not essential for feed degradation and survival of the host (4), it is believed that they contribute to overall gut function, for example, by adding degradative complexity (5), by their ability to scavenge oxygen (6), or by their grazing behavior, which helps to shape and regulate prokaryotic populations (4,7,8). During fermentation of ingested plant material, some of the known rumen ciliates release large amounts of hydrogen produced in their hydrogenosomes (9-11), thereby providing ideal conditions for commensal hydrogenotrophic methanogens. As a consequence, the total number of rumen ciliate protozoa as well as ciliate community composition appears to influence the amount of methane emitted by the host (12-14). However, studies so far have used limited numbers of animals to analyze the impact of ciliate community structure or the roles of individual members of ciliate commun...