Mesophyll conductance to CO2 (gm) limits the diffusion of CO2 to the sites of carboxylation, and may respond rapidly (within minutes) to abiotic factors. Using three Eucalyptus species, we tested the rapid response of gm to irradiance under 21% and 1% O2. We used simultaneous measurements of leaf gas exchange and discrimination against 13CO2 with a tuneable diode laser absorption spectrometer. Measurements under 1% O2 were used to limit uncertainties due to 13C–12C fractionation occurring during photorespiration. Switching irradiance from 600 to 200 mmolm–2 s–1 led to a 60% decrease of gm within minutes in all species under both 21% O2 and 1% O2. The gm response to irradiance is unlikely to be a computation artefact since using different values for the parameters of the discrimination model changed the absolute values of gm but did not affect the relative response to irradiance. Simulations showed that possible rapid changes of any parameter were unable to explain the observed variations of gm with irradiance, except for13C–12C fractionation during carboxylation (b), which, in turn, is dependent on the fraction of leaf C assimilated by phospho-enol pyruvate carboxylase (PEPc) (b). gm apparently increased by 30% when O2 was switched from 21% to 1% O2. Again, possible changes of b with O2 could explain this apparent gm response to O2. Nevertheless, large irradiance or O2-induced changes in b would be required to fully explain the observed changes in gm, reinforcing the hypothesis that gm is responsive to irradiance and possibly also to O2.