The influence of a water deficit was investigated on in vitro plantlets from the rubber tree (Hevea brasiliensis) clone PB 260. The Fraction of Transpirable Soil Water (FTSW) was used to identify three levels of water deficit. Transpiration and stomatal conductance decreased for a FTSW of 0.4 while fluorescence appeared more stable. Proline and ascorbate contents increased from the same level of water deficit. Antioxidant enzyme activities were more stable than glutathione reductase activity, which dramatically increased in line with the water deficit to peak at 0.12 UI/mg for a FTSW of 0.2. When plants were rehydrated, enzyme activities decreased. The accumulation of proline for the lowest FTSW suggested water stress. Antioxidant systems did not play a predominant role for this genotype.