Dissimilatory nitrate reduction (denitrification) in subsurface sediments by indigenous microflora was investigated in samples obtained over a range of depths from 0 to 289 m. Denitrifying activity in sediment samples retrieved from similar stratigraphic horizons at four different sites was determined by measuring the accumulation of N2O using the acetylene blockage technique. Denitrification was detected in unamended samples which received only prereduced deionized water at almost all depths in all sediments sampled. The surface sediments showed the highest denitrification activity. In the deeper sediments, denitrifying activity was much higher in saturated sandy samples and lower or absent in drier clay samples. Addition of nitrate enhanced denitrification activity in all samples from below the water table down to the maximum depth sampled (289 m), while addition of a carbon (succinate) source in general had no stimulatory effect. These results show that denitrifying microorganisms were present in all of the deep subsurface sediments tested in this study. Furthermore, these results suggest that adequate supplies of metabolizable organic carbon were available to support denitrifying activity. However, denitrification may be limited by inadequate supplies of nitrate in the sediments.