The microeukaryotic community in Zodletone Spring, a predominantly anaerobic sulfide and sulfur-rich spring, was examined using an 18S rRNA gene cloning and sequencing approach. The majority of the 288 clones sequenced from three different locations at Zodletone Spring belonged to the Stramenopiles, Alveolata, and Fungi, with members of the phylum Cercozoa, order Diplomonadida, and family Jakobidae representing a minor fraction of the clone library. No sequences suggesting the presence of novel kingdom level diversity were detected in any of the three libraries. A large fraction of stramenopile clones encountered were monophyletic with either members of the genus Cafeteria (order Bicosoecida) or members of the order Labyrinthulida (slime nets), both of which have so far been encountered mainly in marine habitats. The majority of the observed fungal clone sequences belonged to the ascomycetous yeasts (order Saccharomycetales), were closely related to yeast genera within the Hymenobasidiomycetes (phylum Basidiomycetes), or formed a novel fungal lineage with several previously published or database-deposited clones. To determine whether the unexpected abundance of fungal sequences in Zodletone Spring clone libraries represents a general pattern in anaerobic habitats, we generated three clone libraries from three different anaerobic settings (anaerobic sewage digester, pond sediment, and hydrocarbon-exposed aquifer sediments) and partially sequenced 210 of these clones. Phylogenetic analysis indicated that clone sequences belonging to the kingdom Fungi represent a significant fraction of all three clone libraries, an observation confirmed by phospholipid fatty acid and ergosterol analysis. Overall, this work reveals an unexpected abundance of Fungi in anaerobic habitats, describes a novel, yet-uncultured group of Fungi that appears to be widespread in anaerobic habitats, and indicates that several of the previously considered marine protists could also occur in nonmarine habitats.The utilization of culture-independent approaches for describing microbial assemblages in various ecosystems has altered our view on the breadth of prokaryotic diversity. For example, by extensive 16S rRNA gene-based analysis of various marine and terrestrial ecosystems, the number of bacterial divisions and candidate divisions has increased from 12 in 1987 (68) to at least 52 (53). Similarly, numerous novel archaeal lineages, many of which have a global distribution, have been detected in both marine (11) and terrestrial (7) ecosystems.Recently, a similar approach utilizing small subunit (SSU) rRNA gene amplification, cloning, and sequencing has been applied to study eukaryotic diversity. The few available studies primarily examined the microeukaryotic community in different marine environments, including the photic and aphotic zones of pelagic oceans (12,13,25,39,43,61), anoxic marine salt marsh sediments (60), and hydrothermal vents (17,38). Collectively, these studies suggest an unexpected level of diversity with the detection of sequen...