Methanogenesis, sulfate reduction, and rates of carbon mineralization were determined for samples derived at different depths from four cores drilled at the Savannah River Plant, Aiken South Carolina. Three‐gram subsamples of the sediments were dispensed to 10‐mL serum bottles under 5% H2/95% N2and amended with 0.5 mL degassed distilled water with or without the following solutes: formate plus acetate, bicarbonate, lactate, and radiolabeled sulfate, glucose, and Índole. After incubating 1 to 5 days, the sediments were assayed for methane, H2,35S, andI4CO2. No methanogenesis was detected at any depth in any core and sulfate was rarely reduced. Evolution of14CO2from glucose and indole was detected in sediments as deep as 262 and 259 m, respectively. At some depths the14CO2evolution rate was comparable to that of surface soils; however, at other depths no14CO2evolution could be detected. Injection of sterile air into anaerobic incubations increased rates of carbon mineralization at all depths that had demonstrated anaerobic activity and stimulated mineralization activity in sediments that were inactive anaerobically, suggesting a predominance of aerobic metabolism. Increasing the concentration of added glucose and indole often increased the resulting rates of14CO2evolved from these substrates. Our data indicate that both aerobic and anaerobic microorganisms are present and metabolically active in samples from deep subsurface environments.