Aims: To examine the diversity of protozoa in the rumen contents of cow. Methods and Results: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twenty-three clones isolated from rumen fluid fell into two genera identified as Entodinium (69.6% of clones) and Epidinium (31.4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30.4%). Conclusions: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81.1%). Protozoa were not detected in the rumen epithelium. Significance and Impact of the Study: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.