Mesophyll conductance (gm) generally correlates with photosynthetic capacity, although the causal relationship between the two is unclear. The response of gm to various CO2 regimes was measured to determine its relationship to environmental changes that affect photosynthesis. The overall effect of CO2 growth environment on gm was species and experiment dependent. The data did not statistically differ from the previously shown A–gm relationship and was unaffected by CO2 treatment. The consequences of the CO2 effect on gm for interpreting photosynthesis in individual cases were investigated. Substantial effects of assumed versus calculated gm on leaf properties estimated from gas-exchange measurements were found. This differential error resulted in an underestimation in ratio of maximum carboxylation to electron transport, especially in plants with high photosynthetic capacity. Including gm in the calculations also improved the agreement between maximum carboxylation rates and in vitro Rubisco measurements. It is concluded that gm is finite and varies with photosynthetic capacity. Including gm when calculating photosynthesis parameters from gas-exchange data will avoid systematic errors.