Pineapple producing countries lack good quality propagation material to expand cultivars into new areas. Previously, research protocols may increase the offer of high quality plantlets and speed the introduction of new pineapple cultivars. The present work is to evaluate the morpho-physiological changes in plantlets of pineapple [Ananas comosus (L.) Merr. 'MD-2'] during the acclimatization phase. Plantlets were acclimatized under 80% relative humidity, 25.5 °C temperature and photosynthetic photon flux of 400-500 µmol m-2 s-1 as average for 45 d under natural photoperiods. All measurements (plant length, number of leaves and roots, fresh weight, width and length of leaf 'D', net photosynthesis and total transpiration rate) were carried out at the end of in vitro rooting phase coincident with 0 d of acclimatization and at 15, 30 and 45 d thereafter. Photosynthetic activity of in vitro plantlets did not increase during the first 30 d of the acclimatization phase. After 30 d, photosynthetic activity ranged from 5.72 to 9.36 µmol CO2 m-2 s-1 while total transpiration ranged from 6.0 to 1.42 mmol H2O m-2 s-1. During the first 30 days there were no significant differences in number of leaves, length or width of the longest ('D') leaf (cm) or plant length (cm). However, after 45 days plant fresh weight (g), length and width of the 'D' leaf (cm) and root number all increased significantly, while transpiration (mmol H2O m-2 s-1) declined. There were small but significant decreases in chlorophyll a and b (µg g-1 mf.). Increased photosynthetic activity after 30 d shows that the increase in light intensity and the reduction of relative humidity during acclimatization did not constitute inhibitory factors.