Current studies show that a diverse community of microorganisms are capable of growth and activity in the permafrost. These microbial polyextremophiles have developed adaptations allowing them to survive oligotrophic conditions, subzero temperatures, low water availability, high salinity, and background radiation. However, permafrost age appears to have a significant impact on microbial communities, dramatically reducing microbial diversity and biomass. This observation is directly relevant to the use of Earth's permafrost as an analogue for Martian permafrost–which may be billions of years old–and to understanding how life survived during 'snowball Earth' eras, which may have lasted for 6-12 million years. In this experimental research plan, we propose to characterize microbial communities along a permafrost age gradient (5kyr-135kyr) using an interdisciplinary approach. We intend to link microbial phylogeny and function (generated using next-generation sequencing technologies), measurements of microbial activity, and detailed soil physical and chemical data to elucidate the phylogeny, physiology, genes, and pathways that enable microbial survival in permafrost over geological time. Specifically, we propose to: 1) Sample a permafrost geochronosequence from permafrost soils ranging in age from 5kyr-135kyr. 2) Characterize soil physiochemical properties of collected samples. 3) Characterize and compare the taxonomic and functional diversity of microbial permafrost communities using high-throughput sequencing. 4) Characterize and differentiate between active, dead, and dormant microorganisms by combining stable isotope probing, cell sorting, and high-throughput sequencing. 5) Integrate information generated in objectives 1-3 to understand how permafrost age and associated soil properties shape the composition and functional potential of microbial communities. The proposed work has important implications for the field of Exobiology. Although there are an increasing number of metagenomic studies characterizing permafrost microbial communities, none target samples that are older than 6kyr. Our interdisciplinary approach will address this knowledge gap, enabling us to understand the potential of life to adapt to ancient cryoenvironments, the phylogeny and physiology of long-term survivors, and the genes and pathways that are important for long-term survival. These results are directly relevant for establishing the limits of life on Earth and will aid in the search for life elsewhere in the Universe.