When dry soils are rewetted a pulse of CO2 is invariably released, and whilst this phenomenon has been studied for decades, the precise origins of this CO2 remain obscure. We postulate that it could be of chemical (i.e. via abiotic pathways), biochemical (via free enzymes) or biological (via intact cells) origin. To elucidate the relative contributions of the pathways, dry soils were either sterilised (double autoclaving) or treated with solutions of inhibitors (15% trichloroacetic acid or 1% silver nitrate) targeting the different modes. The rapidity of CO2 release from the soils after the drying:rewetting (DRW) cycle was remarkable, with maximal rates of evolution within 6 min, and 41% of the total efflux over 96 h released within the first 24 h. The complete cessation of CO2 eflux following sterilisation showed there was no abiotic (dissolution of carbonates) contribution to the CO2 release on rewetting, and clear evidence for an organismal or biochemical basis to the flush. Rehydration in the presence of inhibitors indicated that there were approximately equal contributions from biochemical (outside membranes) and organismal (inside membranes) sources within the first 24 h after rewetting. This suggests that some of the flux was derived from microbial respiration, whilst the remainder was a consequence of enzyme activity, possibly through remnant respiratory pathways in the debris of dead cells.