Subsurface sediment samples, collected from three boreholes ranging in depths from 0.1 to 260 m, were used in substrate mineralization studies to examine the aerobic metabolic potential of microbial populations indigenous to the deep subsurface. Mineralization was measured by quantifying the amount of14CO2released from radiolabeled acetate, phenol, or 4‐methoxybenzoate added to subsurface sediments at 10μg g‐1. Mineralization of the three compounds was observed in all but a few of the subsurface samples and did not decrease with depth. In addition, mineralization data collected from similar geologic formations from the different boreholes indicated that there was significant lateral continuity of microbial activity. Regression analyses were performed to determine which environmental factors were related to microbial metabolic potential. Mineralization was positively correlated with heterotrophic abundance as measured by plate counts. Other parameters that appeared to influence metabolic potential included pH and clay content.