Theoretical models of photosynthetic isotopic discrimination of CO2 (13C and 18O) are commonly used to estimate mesophyll conductance (gm). This requires making simplifying assumptions and assigning parameter values so that gm can be solved for as the residual term. Uncertainties in gm estimation occur due to measurement noise and assumptions not holding, including parameter uncertainty and model parametrization. Uncertainties in the 13C model have been explored previously, but there has been little testing undertaken to determine the reliability of gm estimates from the 18O model (gm18). In this study, we exploited the action of carbonic anhydrase in equilibrating CO2 with leaf water and manipulated the observed photosynthetic discrimination (18O) by changing the oxygen isotopic composition of the source gas CO2 and water vapor. We developed a two-source 18O method, whereby two measurements of 18O were obtained for a leaf with its gas-exchange characteristics otherwise unchanged. Measurements were performed in broad bean (Vicia faba) and Algerian oak (Quercus canariensis) in response to light and vapor pressure deficit. Despite manipulating the 18O by over 100, in most cases we observed consistency in the calculated gm18, providing confidence in the measurements and model theory. Where there were differences in gm18 estimates between source-gas measurements, we explored uncertainty associated with two model assumptions (the isotopic composition of water at the sites of CO2-water exchange, and the humidity of the leaf internal airspace) and found evidence for both. Finally, we provide experimental guidelines to minimize the sensitivity of gm18 estimates to measurement errors. The two-source 18O method offers a flexible tool for model parameterization and provides an opportunity to refine our understanding of leaf water and CO2 fluxes.