Acclimatization of sugarcane plantlets (Saccharum spp. hybrid) var. C91-301, micropropagated in temporary immersion bioreactors, was studied. When in vitro plantlets were transferred to acclimatization conditions, photosynthesis increased only slightly during the first 7 d. After this period, the increase was constant with only a small decline after transfer to uncontrolled external conditions. Production of fresh and dry weights, number of leaves and roots, leaf area, stomatal density and chlorophyll contents were evaluated during the acclimatization period. From day 14 on, sugarcane plantlets exhibited low but positive photosynthesis due to the new formation of leaves and roots. The increase in light intensity and the gradual reduction of relative humidity during acclimatization did not constitute inhibitory factors, as can be interpreted from the increased photosynthetic activity until 21 d. Stomatal density and leaf area after 21 d were also increased, while the chlorophyll content declined.