A transient increase in soil carbon dioxide efflux after rewetting of previously dry soils, termed the Birch effect, can significantly influence the ecosystem carbon balance. This has generally been related to increased soil microbial respiration in response to a temporal increase in labile soil carbon. In order to quantify ecosystem carbon losses by the Birch effect and to trace the underlying biogeochemical processes, we monitored the effects of first natural rain pulses on soil and ecosystem carbon dioxide fluxes and their isotopic composition (δ13C) after an extended summer drought in a Mediterranean oak woodland. While GPP was affected negatively, rain largely enhanced soil respiratory processes, which increased their relative contribution to NEE by up to 95%, resulting in a significant net carbon loss from the ecosystem. This was reflected by high correlation (r 0.91) between the two fluxes. Further, isotopic composition of soil respiration (δ13CS) explained on average 71% of the isotopic composition of ecosystem respiration (δ13CR). We found a strong relationship between soil moisture and the increase in soil respiration (RS) and NEE, indicating that the Birch effect observed during this study did not result from a transient increase in labile soil carbon but from a gradual (several days) moisture response of size and/or activity of the soil microbial community. We also observed large variation in δ13C of soil and ecosystem respired CO2 that corresponded to the rain pulses with enrichment of up to 8 and 6‰, respectively, and a subsequent depletion to initial values during the following dry days, which might be explained by increased relative contribution of soil microbial communities from deeper soil layers to overall soil respiration, a switch in the respired carbon source (e.g. anaplerotic carbon) and changes in apparent fractionation during the mineralization of soil organic matter after the depletion of labile carbon pools.