In C(3) plants, diffusion of CO(2) into leaves is restricted by stomata and subsequently by the intercellular airspaces and liquid phase into chloroplasts. While considerable information exists on the effect of environmental conditions on stomatal conductance (g(s)), little is known on whether the mesophyll conductance to CO(2) diffusion (g(m)) changes with respect to photon flux density (PFD) and CO(2) partial pressure (pCO(2)). In this study, the effects of PFD and/or pCO(2) on g(m) were examined in wheat leaves by combining gas exchange with carbon isotope discrimination measurements using a membrane inlet mass spectrometer. Measurements were made in 2% O(2) to reduce the fractionation associated with photorespiration. The magnitude of g(m) was estimated using the observed carbon isotope discrimination (Delta), ambient and intercellular pCO(2), CO(2) assimilation and respiration rates, either from an individual measurement made under one environmental condition or from a global fit to multiple measurements where PFD was varied. It was found that respiration made a significant and variable contribution to the observed discrimination, which associated with the difference in isotopic composition between CO(2) in the greenhouse and that used for gas exchange measurements. In wheat, g(m) was independent of PFD between 200 and 1500 micromol m(-2) s(-1) and was independent of p(i) between 80 and 500 microbar.