A vast subsurface biosphere exists beneath the seafloor, but there is little understanding of exactly how microbes affect chemical exchange between seawater and oceanic basement, or how this affects global geochemical budgets. Recent research shows that microbes have an active role in altering the volcanic rocks on the seafloor, and new isotope techniques have been developed that can tell the temperatures at which the alteration has taken place. This research uses the stable isotopes of sulfur and oxygen in altered seafloor mafic lavas sampled by NSF-funded Ocean Drilling Program to (1) trace the distribution of the subsurface biosphere in the oceanic basement, (2) understand how microbial processes affect chemical and isotope exchange between seawater and the crust, and (3) determine the influence of microbial processes on geochemical cycling in the oceans. This is the first study of its kind, and as such is potentially transformative in terms of its ability to inform us about life in the deep biosphere. Research will involve the sulfur contents and isotopes in rocks from cool fluid downwelling sites along the flanks of mid-ocean ridges. The oxygen isotope compositions of phosphate adsorbed to iron-oxyhydroxides from warm and cool sites will also be analyzed to examine the effects of in-situ biological activity and estimate the temperature at which microbial metabolic processes are occurring, giving an idea of the depth in the ocean crust to which life extends. The broader impacts of the work include support of an African American female researcher and the training of undergraduates and a postdoc in state-of-the-art stable isotope techniques. Minority high school students in the New Haven CT area will also be engaged in the laboratory research. In addition, public outreach associated with the project will be carried out by the Peabody Museum in New Haven and the University of Michigan Nano-Rocks summer high school camp. The work will incorporate research results into courses and support analytical facilities at both institutions.