Leafy shoots excised from rain tree (Samanea saman Merr.) plantlets in vitro were cultured for 90 days on vermiculite containing basic Murashige and Skoog (1962) solution containing 0 (sugar-free), 29, 58 or 88 mM (30 g L−1) sucrose in a 240-mL naturally ventilated vessel with the number of air exchanges being 5.1 h−1 at CO2 concentration in a plant growth chamber of 1000 μmol mol−1. Photosynthetic photon flux (PPF), relative humidity and air temperature on the culture shelf were, respectively, 100 μmol m−2 s−1, 45% and 25 °C. Averages of fresh and dry weights, stem and primary root lengths, leaf area and number of unfolded leaves, primary and lateral roots of the plantlets on day 90 were greatest with no sucrose in the medium, followed by 29, 58 and 88 mM sucrose in the medium. Net photosynthetic rate and chlorophyll concentration of the plantlets on day 90 increased with decreasing sucrose concentration in the medium. CO2 concentrations in the vessel on day 90 at sucrose concentrations of 0, 29, 58 and 88 mM in the medium were, respectively, 362, 679, 795 and 880 μmol mol−1due to the higher net photosynthetic rates of plantlets at lower sucrose concentrations in the medium. It is considered that the photosynthesis and growth of in vitro plantlets and their subsequent growth after transfer to the greenhouse could be further promoted if they were cultured in vitro at a CO2 concentration higher than 1000 μmol mol−1 and a PPF higher than 100 μmol m−2 s−1 in the culture room.