Internal conductance to carbon dioxide is a key aspect of leaf photosynthesis although is still not well understood. It is thought that it comprises two components, namely, a gas phase component (diffusion from intercellular spaces to cell walls) and a liquid phase component (dissolution, diffusion in water, hydration equilibrium). Here we use heavy water (D₂O), which is known to slow down CO₂ hydration by a factor of nearly three. Using ¹²C/¹³C stable isotope techniques and Xanthium strumarium L. leaves, we show that the on-line carbon isotope discrimination (Δ¹³C, or Δ_obs) associated with photosynthesis is not significantly decreased by heavy water, and that the internal conductance, estimated with relationships involving the deviation of Δ¹³C, decreased by 8–40% in 21% O₂. It is concluded that in typical conditions, the CO₂-hydration equilibrium does not exert an effect on CO₂ assimilation larger than 9%. The carbon isotope discrimination associated with CO₂ addition to ribulose-1,5,bisphosphate by Rubisco is slightly decreased by heavy water. This effect is proposed to originate from the use of solvent-derived proton/deuteron during the last step of the catalytic cycle of the enzyme (hydration/cleavage).