Strawberry plantlets (Fragaria X ananassa Duch. cv. Kent) were submitted to a factorial arrangement of 2 photosynthetic photon fluxes (PPF) (80 and 150 μmol·m-2·s-1, PAR) and 2 CO2 concentrations (330 and 3000 ppm) during the in vitro rooting stage. Leaves were tagged and placed in a growth chamber tor acclimatization. Photosynthetic capability of leaves from different origins was determined by measuring the initial and total activity of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco), but the contribution of Phosphoenolpyruvate carboxylase (PEPCase) to fixation was also examined High CO2 concentration and PPF significantly increased fresh weight and surface area in vitro and after 4 weeks ex vitro. Improved growth was not the result of increased autotrophy in vitro since initial rubisco activity was 10 times lower than that of de novo formed leaves and declined under high CO2 and PPF. Carbon dioxide concentration and PPF had no effect on total activity of rubisco. Low activation state and total activity of rubisco in in vitro leaves is the cause of poor photosynthetic activity in vitro Persistent in vitro leaves after 4 weeks of acclimatization did not have higher total activity of rubisco, but the activation state was 4 times larger than the corresponding activity in vitro which might thus provide for non-negligible contribution to photosynthetic carbon assimilation. The possible inhibition of photosynthesis by the presence of sugar in the medium is discussed.