The goal of this project was to explore the mechanisms of subsurface organoheterotrophy by identifying the range of extracellular peptidases present in sediments of the White Oak River, NC, consistent with C-DEBI Research Theme 1, Activity in the Deep Subseafloor Biosphere: function & rates of global biogeochemical processes. This grant funded two sampling expeditions to the White Oak River as well as extensive laboratory work with a purified peptidase that was supplied by collaborators Andrzej Joachimiak and Karolina Michalska of Argonne National Laboratory and preparatory work on peptidases of the Tennessee River. So far this dataset has led to the submission of two manuscripts, with one more manuscript in preparation. The White Oak River work showed that a wide range of peptidases are present in depths up to 80cm in the White Oak River, which is deeper than the zone of methanogenesis. Although absolute peptidase activities declined with depth, activities normalized to cell abundance were roughly constant, and activities normalized to organic carbon oxidation rates increased nearly two orders of magnitude relative to the surface, indicating that extracellular peptidases were important to the subsurface ecosystem. Biochemical analysis of a purified peptidase that was expressed by the Argonne group showed it to be a novel aminopeptidase with specificity for N-terminal cysteine, a function not previously observed in peptidses. In summary, in situ and in vitro studies of subsurface peptidases revealed that they are ecologically important and may contain novel properties.