The degree to which increased soil respiration rates following wetting is caused by plant (autotrophic) versus microbial (heterotrophic) processes, is still largely uninvestigated. Incubation studies suggest microbial processes play a role but it remains unclear whether there is a stimulation of the microbial population as a whole or an increase in the importance of specific substrates that become available with wetting of the soil. We took advantage of an ongoing manipulation of leaf litter 14C contents at the Oak Ridge Reservation, Oak Ridge, Tennessee, to (1) determine the degree to which an increase in soil respiration rates that accompanied wetting of litter and soil, following a short period of drought, could be explained by heterotrophic contributions; and (2) investigate the potential causes of increased heterotrophic respiration in incubated litter and 0–5 cm mineral soil. The contribution of leaf litter decomposition increased from 6 ± 3 mg C m−2 hr−1 during a transient drought, to 63 ± 18 mg C m−2 hr−1 immediately after water addition, corresponding to an increase in the contribution to soil respiration from 5 ± 2% to 37 ± 8%. The increased relative contribution was sufficient to explain all of the observed increase in soil respiration for this one wetting event in the late growing season. Temperature (13°C versus 25°C) and moisture (dry versus field capacity) conditions did not change the relative contributions of different decomposition substrates in incubations, suggesting that more slowly cycling C has at least the same sensitivity to decomposition as faster cycling organic C at the temperature and moisture conditions studied.