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The oceanic waters below a depth of 200 m represent, in terms of volume, the largest habitat of the biosphere, harboring approximately 70% of the prokaryotic biomass in the oceanic water column. These waters are characterized by low temperature, increasing hydrostatic pressure, and decreasing organic matter supply with depth. Recent methodological advances in microbial oceanography have refined our view of the ecology of prokaryotes in the dark ocean. Here, we review the ecology of prokaryotes of the dark ocean, present data on the biomass distribution and heterotrophic and chemolithoautotrophic prokaryotic production in the major oceanic basins, and highlight the phylogenetic and functional diversity of this part of the ocean. We describe the connectivity of surface and deep-water prokaryotes and the molecular adaptations of piezophilic prokaryotes to high hydrostatic pressure. We also highlight knowledge gaps in the ecology of the dark ocean's prokaryotes and their role in the biogeochemical cycles in the largest habitat of the biosphere.