Chlorophyll a fluorescence kinetics, net photosynthetic rate (PN), water relations, and photosynthetic pigment contents were studied during acclimation of in vitro grown tobacco to higher irradiance (HL; 700 μmol m-2 s-1). Plantlets were grown on medium containing sucrose in glass vessels (G-plants) or in Magenta boxes (M-plants) with better CO2 supply in the latter ones. The effect of HL was studied either (1) in plantlets grown under original in vitro conditions (closed vessels), (2) in in vitro plantlets exposed to ambient CO2 concentration (covers removed), or (3) in plantlets transplanted to ex vitro into pots with sand and nutrient solution. Higher PN, and fraction of closed photosystem 2 (PS2) centres (1 - qP), and lower content of xanthophyll cycle pigments were found in M-plants compared to G-plants. HL treatment caused photoinhibition particularly in plants kept in closed vessels. This was indicated by the decrease in the ratio of Fv/Fm and by the increase in non-photochemical quenching, 1 - qp, and content of xanthophyll cycle pigments. Better CO2 supply ensured by the removal of closure lead to the moderate reduction of symptoms of photoinhibition, although stomatal conductance (gs), transpiration rate (E), and PN were negatively affected. The main reason was the decrease in relative air humidity, which caused similar reduction of PN, E, and gs after the transfer of plantlets to ex vitro. Nevertheless, plant response to HL seemed not to be affected by any possible root injury caused by transfer to ex vitro. The differences in contents of xanthophyll cycle pigments, degree of de-epoxidation, PN, and quenching parameters between M- and G-plantlets were still significant 7 d after ex vitro transfer and HL acclimation. and Š. Semorádová, H. Synková, J. Pospíšilová.