Microbial life on Earth is pervasive, inhabiting even the most extreme surface environments and extending deep into the subsurface. Because of the limited impact of any individual microbe, much of what is known about microbial activity in the environment has been inferred from chemical changes, phylogenetic similarity to lab organisms, or proxy records. It is now firmly established that microbial processes drive many geochemical cycles on Earth, but little is known about the activity and impact of the deep subsurface biosphere on these processes. A number of approaches have been used to investigate the subsurface biosphere including chemical profiling, DNA and RNA sequencing, and, in a limited number of settings, lipid biomarkers. Lipids are ubiquitous in biology, deriving from the cellular membrane, and have the potential to be preserved for billions of years in sedimentary environments. Lipid biomarkers are used as proxy records for the presence and activity of particular microbes, and have yielded rich records of microbial activity in modern and ancient environments. For this reason, the search for life beyond the Earth includes the search for extraterrestrial lipid biomarkers. However, biomarker proxies require calibration to the environment of interest, and thus we must first ask what sort of biomarker patterns (both structural and isotopic) might be expected in the Martian or Europan subsurface. Characterization of the biomarker profiles of in situ microbes from the terrestrial subsurface (an analog environment to extraterrestrial targets) will significantly advance the application of these powerful tools. In this proposal we present a plan for detailed investigation of the lipid biomarkers present in the deep continental subsurface. A single lipid contains multiple layers of information useful for biogeochemical interpretation, including the polar head group (for living microbes), the structure of the hydrocarbon skeleton (both living and fossil), and the isotopic composition of the hydrocarbon core (both living and fossil). We will analyze all three of these components from a range of terrestrial locations with the aim of identifying signatures common to the subsurface. Furthermore, we will apply the same methods to isolated subsurface organisms to 1) advance understanding of the biological controls in biomarkers patterns and 2) serve as calibration points for our environmental investigations. A unique partnership between this proposal and the NAI Life Underground will provide access to samples, geochemical data, molecular data, and isolated subsurface organisms well beyond what would be possible alone. The methodology used in this study is established in the community and available in the laboratories of the principle investigator and collaborators. Measurements of intact polar lipids will be made by via high-pressure liquid chromatography coupled to tandem mass spectrometry. Measurements of core lipids for structural and isotopic composition will be preformed using gas chromatography coupled to mass spectrometry in the laboratory of PI Osburn. This proposal will directly advance goals of the NASA astrobiology roadmap (NAR) and ROSES 2014 Exobiology NRA. Characterizing the biomarker fingerprint of the deep subsurface biosphere is a critical first step towards understanding signatures from extraterrestrial targets, directly related to NAR Goal 7, Determine how to recognize signatures of life on other worlds and on early Earth. Furthermore, the NRA research emphasis, Biosignatures and Life Elsewhere specifically solicits for biosignature studies of analog environments, in this case, the deep continental subsurface. In addition, the environmental extremes present in the deep subsurface are drivers of adaptation and evolution thus study of these systems directly addresses NAR Goal 5, Objective 5.3 – Biochemical adaptation to extreme environments and NRA research emphasis, Early Evolution of Life and the Biosphere.