The specificity factor (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) defines the relative rates of carboxylation and oxygenation of Ribulose-1,5-bisphosphate (RuBP) catalysed by the enzyme. The determination of S for Rubisco purified from the Vitis vinifera L. cvs Tempranillo and Manto Negro is described here for the first time. Rubisco extraction was made in Bicine buffer with the inclusion of polyethylene glycol (PEG), β-mercaptoethanol, diethyldithio-carbamic acid (DIECA) and several protease inhibitors. Furthermore, in the same cultivars, the apparent in vivo specificity factor for Rubisco (S*), was obtained from gas exchange and chlorophyll fluorescence measurements. For both cultivars the values of S were close to 100 at 25°C. However, in mature leaves, S* was about 67 for Manto Negro and 55 or 77 for Tempranillo, depending on leaf age. Leaves of plants under drought showed even lower values. These discrepancies between S and S* are ascribed to equating CO₂ in the sub-stomatal cavity with CO₂ at the Rubisco catalytic site in the chloroplast. However, S* values from young developing leaves were very close to S for both cultivars. It is concluded that estimations of S* based gas exchange and chlorophyll fluorescence data are reliable only in thin, young and non-stressed leaves.