Microbial activity in saturated, subsurface sediments in riparian forests may be supported by recent photosynthate or ancient (>500 ybp) soil organic carbon (SOC) in buried horizons. Metabolism of ancient SOC may be particularly important in riparian zones, considered denitrification hot spots, because denitrification in the riparian subsurface is often C-limited, because buried horizons intersect deep flow paths, and because low C mineralization rates can support ecosystem-relevant rates of denitrification. Buried horizons are common where alluvial processes (stream migration, overbank flow) have dominated riparian evolution. Our objectives were to determine: (1) the extent to which ancient SOC directly supports subsurface microbial activity; (2) whether different C sources support microbial activity in alluvial versus glaciofluvial riparian zones; and (3) how microbial use of ancient SOC varies with depth. In situ groundwater incubations and 14C dating of dissolved inorganic carbon revealed that ancient SOC mineralization was common, and that it constituted 31–100% of C mineralization 2.6 m deep at one site, at rates sufficient to influence landscape N budgets. Our data failed to reveal consistent spatial patterns of microbially available ancient C. Although mineralized C age increased with depth at one alluvial site, we observed ancient C metabolism 150 cm deep at a glaciofluvial site, suggesting that subsurface microbial activity in riparian zones does not vary systematically between alluvial and glaciofluvial hydrogeologic settings. These findings underscore the relevance of ancient C to contemporary ecosystem processes and the challenge of using mappable surface features to identify subsurface ecosystem characteristics or riparian zone N-sink strength.