A study of two grapevine cultivars, Vitis vinifera cv. Chardonnay and cv. Merlot revealed there were differences in photosynthesis. A more extensive comparative study of photosynthesis of these two cultivars in relation to internal CO2 (Ci) concentration at leaf temperatures from 15 to 45 °C was, therefore, undertaken to explain these differences. Average rates of photosynthesis at ambient and saturating CO2 concentrations were confirmed to be higher in Merlot leaves across all leaf temperatures. The physiological explanation was partially attributable to much higher stomatal conductances at lower temperatures and by much higher rates of ribulose 1, 5-bisphosphate (RuBP) regeneration (Jmax) at all temperatures in Merlot leaves. By contrast, apparent rates of RuBP carboxylation did not vary between the cultivars except above 40 °C where rates were highest in the Merlot leaves. There were cultivar differences in temperature optima for assimilation (25 and 30 °C), but not for Vcmax (44, +45 °C) but some differences in Jmax (37 and 39 °C). The transition from RuBP regeneration limited to RuBP carboxylation-limited assimilation occurred at about 34 °C in both cultivars, suggesting the high temperatures may have been detrimental to assimilation. Although the stomatal limitation to assimilation increased with increasing leaf temperature, there were no cultivar differences in the temperature dependency of the stomatal limitation to assimilation. Thus, the confirmed differences in assimilation in Merlot compared to Chardonnay leaves were concluded to be a result of stomatal conductance and an inherently greater capacity for RuBP regeneration.