Although a significant amount of the organic C stored in soil resides in subsurface horizons, the dynamics of subsurface C stores are not well understood. The objective of this study was to determine if changes in soil moisture, temperature, and nutrient levels have similar effects on the mineralization of surface (0–25 cm) and subsurface (below 25 cm) C stores. Samples were collected from a 2 m deep unsaturated mollisol profile located near Santa Barbara, CA, USA. In a series of experiments, we measured the influence of nutrient additions (N and P), soil temperature (10–35°C), and soil water potential (−0.5 to −10 MPa) on the microbial mineralization of native soil organic C. Surface and subsurface soils were slightly different with respect to the effects of water potential on microbial CO2 production; C mineralization rates in surface soils were more affected by conditions of moderate drought than rates in subsurface soils. With respect to the effects of soil temperature and nutrient levels on C mineralization rates, subsurface horizons were significantly more sensitive to increases in temperature or nutrient availability than surface horizons. The mean Q10 value for C mineralization rates was 3.0 in surface horizons and 3.9 in subsurface horizons. The addition of either N or P had negligible effects on microbial CO2 production in surface soil layers; in the subsurface horizons, the addition of either N or P increased CO2 production by up to 450% relative to the control. The results of these experiments suggest that alterations of the soil environment may have different effects on CO2 production through the profile and that the mineralization of subsurface C stores may be particularly susceptible to increases in temperature or nutrient inputs to soil.