Drying and wetting cycles of O horizon in forest soils have not received much attention, partly due to methodological limitations for nondestructive monitoring of the O horizon water content. The objective of this study was to determine the importance of moisture limitations in the O horizon of a temperate forest on summertime soil respiration. We measured soil respiration in three replicated plots in a mixed deciduous forest at Harvard Forest, Massachusetts, weekly from May to October 2001. Direct Current (DC) half-bridge sensors that had been calibrated using destructive samples of the Oi and Oe/ Oa horizons were placed in the Oi and Oe/Oa horizons to record hourly changes of gravimetric water contents. Soil temperature explained 47% of the variation in soil respiration using the Arrhenius equation. The residuals of the temperature model were linearly correlated with gravimetric water content of the Oi horizon (r2 = 0.72, P < 0.0001) and Oe/Oa horizon (r2 = 0.56, P < 0.001), indicating that temporal variation in soil respiration can be partly explained by water content of the O horizon. Additionally, a laboratory study was performed to evaluate drying/wetting cycles of the O horizon at constant temperature. Even small simulated rainfall amounts of 0.5 mm significantly increase CO2 flux from dry O horizon within a few minutes. The duration of CO2 pulses increased with the amount of applied water, lasting from a few hours to days. A strong correlation between CO2 release and water content of the O horizons demonstrates a clear regulatory role of litter water content on decomposition within the O horizons.