Cold seeps are areas of the seafloor where hydrogen sulfide- and methane-rich fluid seepage occurs, often sustaining chemosynthetic ecosystems. It is well known that both archaea and bacteria oxidize sulfides and methane to produce chemical energy and that several endemic animals use this energy to thrive in cold seeps. On the other hand, there is little knowledge regarding diversity and ecology of microbial eukaryotes in this ecosystem. In this study we isolated environmental RNA and DNA from microbial mats of cold-seep sediment in Sagami Bay, Japan, and retrieved eukaryotic small-subunit ribosomal RNA sequences with polymerase chain reaction methods followed by clone library construction. Most RNA-derived clones obtained were from ciliates, although DNA-derived clones were mainly from the fungus Cryptococcus curvatus, suggesting that ciliates are active in the environment. The ciliate sequences were phylogenetically diverse, and represented eight known class lineages as well as undesignated lineages. Because most ciliates are bacterivorous, it is highly likely that the ciliates for which sequences were recovered play a role in the food web of this ecosystem as grazers of microbial mats. In addition, given that the environment studied is under highly reduced (anoxic) conditions, based on the prokaryotic community structure deduced from T-RFLP profiles, the ciliates detected may be obligatory or facultative anaerobes.