Desiccation after ex vitro transfer is one of the main causes of mortality of tissue culture-derived plantlets. Water loss and chlorophyll a fluorescence parameters were determined in micropropagated regenerants derived from a superior 80-year-old black mulberry (Morus nigra L.) tree in the course of a 56-day ex vitro acclimatization period. On days 0, 7 and 35 after transfer, leaves that were formed in vitro lost more than 80% of their water content within 90 min of air-drying. Poor control of water loss clearly demonstrated the inability of non-acclimatized plantlets to avoid desiccation. In contrast, ex vitro formed leaves sampled on day 56 showed slight wilting and had a water loss rate of less than 29% after 90 min. The lowest rates of maximal photochemical efficiency of photosystem II (F_v/F_m), potential electron capacity ('area'), and maximum-to-initial fluorescence ratio (F_m/F₀) were found on day 0 at the end of the in vitro culture period. The recovery of these rates took place gradually during the acclimatization period with the most prominent changes found between the days 0 and 7 as well as 35 and 56. The acclimatization was achieved 8 weeks after transfer when new ex vitro formed leaves started to grow.