BACKGROUND: The deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we examined the richness of eukaryotic DNA in deep Arctic and Southern Ocean samples using massively parallel sequencing of the 18S ribosomal RNA (rRNA) V9 hypervariable region. In very small volumes of sediments, ranging from 0.35 to 0.7 g, we recovered up to 7,499 unique sequences per sample. By clustering sequences having up to 3 differences, we observed from 942 to 1756 Operational Taxonomic Units (OTUs) per sample. Taxonomic analyses of these OTUs showed that DNA of all major groups of eukaryotes is represented at the deep-sea floor. The dinoflagellates, cercozoans, ciliates, and euglenozoans predominate, contributing to 17%, 16%, 10%, and 8% of all assigned OTUs, respectively. Interestingly, many sequences represent photosynthetic taxa or are similar to those reported from the environmental surveys of surface waters. Moreover, each sample contained from 31 to 71 different metazoan OTUs despite the small sample volume collected. This indicates that a significant faction of the eukaryotic DNA sequences likely do not belong to living organisms, but represent either free, extracellular DNA or remains and resting stages of planktonic species. CONCLUSIONS/SIGNIFICANCE: In view of our study, the deep-sea floor appears as a global DNA repository, which preserves genetic information about organisms living in the sediment, as well as in the water column above it. This information can be used for future monitoring of past and present environmental changes.