Limited information is available on the potential of subsoil and underlying alluvial gravels (vadose zone) to denitrify leached nitrate, though it is recognized that C availability is an important constraint. In this laboratory study, we used samples (<4 tom fraction) from subsoil (0.4-0.85 m depth) and alluvial gravel layers (1.2-4.8 m) derived from a site in Canterbury, New Zealand, to quantify denitrification potential and examine the denitrification response to inputs of dissolved organic C (DOC). As C sources, we used DOC extracted in either cold (20 degrees C) or hot (80 degrees C) water from topsoil and subsoil (assumed to represent C substrates likely to be transported into the subsoil in drainage). Measurements of potential denitrification enzyme activity (DEA, via samples amended with glucose and nitrate-N) showed the alluvial gravel samples had denitrification capability although rates were low (<5 mu g N2O-N kg(-1) h(-1)) compared with those reported for topsoils. The effect of the soil-derived DOC, at addition rates 16-215 mg kg(-1), on denitrification was determined in a 7-d anaerobic incubation using acetylene to block reduction of N2O to N-2. There was a lag of 24-36 h, during which N2O production was low, followed by a period of rapid increase (36-96 h), and a final phase (120-168 h) in which N2O concentrations remained relatively stable. During the rapid phase, the N2O production rate was very high (up to 440-500 mu g N kg(-1) h(-1) at the highest DOC addition rate) and exceeded that measured in the 8-h DEA assay (a measure of indigenous denitrifier enzymes). The post-96 h decline in N2O production rate was attributed to depletion of available C. The temporal pattern of CO2-C production was similar to that of N2O, though the lag period was shorter (12-24 h). The ratio of N2O-N to CO2-C increased with time; the maximum was circa 0.3:1 at the highest addition rate of hot water extractable DOC. Our results suggest that, with input of DOC substrate, alluvial gravel materials could generate enough enzyme during a 1-2 day anaerobic period to denitrify significant quantities of nitrate. However, in situ denitrification in alluvial gravels may be low because of biophysical limitations such as low C inputs of bioavailable C and anaerobicity mostly confined to localized zones or lenses. (C) 2013 Elsevier Ltd. All rights reserved.