The possible existence of functionally active, metabolically diverse microbial ecosystems within the deep seafloor beneath sites of hydrothermal venting (the so-called "subvent biosphere") has been predicted, but is as yet unproved, by a number of interdisciplinary investigations in a variety of hydrothermal systems within different tectonic and geological settings. Integrated Ocean Drilling Program (IODP) Expedition 331, Deep Hot Biosphere, proposes to characterize subseafloor microbial ecosystems within the context of their physical, geochemical, and hydrogeologic setting in the Iheya North hydrothermal field in the mid-Okinawa Trough at three primary drilling sites (INH-4D, INH-5D, and INH-1D) and three contingency sites (INH-1C, INH-3C, and INH-2C). An important objective of this project is to determine the compositional and isotopic shift in biologically essential chemical compounds in the hydrothermal fluids, including carbon dioxide, methane, hydrogen, sulfur/sulfide, ammonia, oxygen, and various organic compounds, which are variably enriched or depleted by physical, chemical, and biological processes throughout the hydrothermal fluid pathways in the mid-Okinawa Trough. These chemical reactants and products support the activity and functions of unique subseafloor microbial communities within different habitats of hydrothermal fluid migration. Extremely high concentrations of dissolved C-bearing gases, mainly CO2 and CH4, in the hydrothermal fluids, their stable carbon isotopic characteristics, potentially widespread fluid reservoirs in the sediments and pyroclastic deposits, and phase separation and phase partition of hydrothermal fluids provide key clues to understanding how the active deep biosphere operates and interacts with subseafloor hydrothermal systems in the mid-Okinawa Trough. Understanding such interactions is one of primary scientific goals identified in the Initial Science Plan for IODP. During the long history of the Deep Sea Drilling Project, the Ocean Drilling Program, and IODP, Expedition 331 will be the first to drill an active hydrothermal system in a sediment-filled backarc rift along a continental margin. The interdisciplinary approach we propose will provide clues to the evolution and early success of primordial microbial ecosystems on Earth, as well as an understanding of the nature of the deep hot biosphere on present-day Earth.