The contribution of photosynthesis and soil respiration to net land atmosphere carbon dioxide (CO2) exchange can be estimated based on the differential influence of leaves and soils on budgets of the oxygen isotope composition (18O) of atmospheric CO2. To do so, the activity of carbonic anhydrases (CAs), a group of enzymes that catalyse the hydration of CO2 in soils and plants, needs to be understood. Measurements of soil CA activity typically involve the inversion of models describing the 18O of CO2 fluxes to solve for the apparent, potentially catalysed, rate of CO2 hydration. This requires information about the 18O of CO2 in isotopic equilibrium with soil water, typically obtained from destructive, depth-resolved sampling and extraction of soil water. In doing so, an assumption is made about the soil water pool that CO2 interacts with, which may bias estimates of CA activity if incorrect. Furthermore, this can represent a significant challenge in data collection given the potential for spatial and temporal variability in the 18O of soil water and limited a priori information with respect to the appropriate sampling resolution and depth. We investigated whether we could circumvent this requirement by inferring the rate of CO2 hydration and the 18O of soil water from the relationship between the 18O of CO2 fluxes and the 18O of CO2 at the soil surface measured at different ambient CO2 conditions. This approach was tested through laboratory incubations of air-dried soils that were re-wetted with three waters of different 18O. Gas exchange measurements were made on these soils to estimate the rate of hydration and the 18O of soil water, followed by soil water extraction to allow for comparison. Estimated rates of CO2 hydration were 6.814.6 times greater than the theoretical uncatalysed rate of hydration, indicating that CA were active in these soils. Importantly, these estimates were not significantly different among water treatments, suggesting that this represents a robust approach to assay the activity of CA in soil. As expected, estimates of the 18O of the soil water that equilibrates with CO2 varied in response to alteration to the 18O of soil water. However, these estimates were consistently more negative than the composition of the soil water extracted by cryogenic vacuum distillation at the end of the gas measurements with differences of up to 3.94 VSMOWSLAP. These offsets suggest that, at least at lower water contents, CO2 H2O isotope equilibration primarily occurs with water pools that are bound to particle surfaces and are depleted in 18O compared to bulk soil water.