Carbonic anhydrase (CA) activity in leaves catalyzes the O-18 exchange between CO2 and water during photosynthesis. This feature has been used to estimate the mesophyll conductance to CO2 (g(m)) from measurements of online (COO)-O-18 photosynthetic discrimination (Delta O-18) Based on CA assays on leaf extracts, it has been argued that CO2 in mesophyll cells should be in isotopic equilibrium with water in most C-3 species as well as many C-4 dicot species. However, this seems incompatible with Delta O-18 data that would indicate a much lower degree of equilibration, especially in C-4 plants under high light intensity. This apparent contradiction is resolved here using a new model of C-3 and C-4 photosynthetic discrimination that includes competition between CO2 hydration and carboxylation and the contribution of respiratory fluxes. This new modeling framework is used to revisit previously published data sets on C-3 and C-4 species, including CA-deficient plants. We conclude that (1) newly Delta O-18-derived g(m) values are usually close but significantly higher (typically 20% and up to 50%) than those derived assuming full equilibration and (2) despite the uncertainty associated with the respiration rate in light, or the water isotope gradient between mesophyll and bundle sheath cells, robust estimates of Delta O-18-derived g(m) can be achieved in both C-3 and C-4 plants.Read less <