Three parameters (i.e. the water content, soluble sugar content and minimal air temperature) can be used to predict the cold acclimation process of walnut trees. In order to test this assumption, two-year-old walnuts were defoliated at two different dates, i.e. mechanical defoliation in early October (early leaf fall, EF) or natural defoliation in early November (natural leaf fall. NF) and conditioned in either outdoor freeze-deprived or cold-deprived (T(min) > 13 degrees C) greenhouses over winter. Even if early defoliation date could have affected short clay signal perception (SDSP), water balance and carbohydrate metabolism were more altered. EF treatment, by stopping transpiration, significantly increased tree's water content and at warm temperature high root activity stopped normal winter dehydration. Starch content decreased in all treatments, but there was only a significant increase in soluble sugar content when water content had sufficiently decreased. Thus, depending on date of defoliation, cold-deprived trees were or were not able to acclimate to frost (minimal frost hardiness = -21.8 degrees C vs. -22.1 degrees C in controls (freeze-deprived) for NF and -13.7 degrees C vs. -25.3 degrees C in controls for EF). Different treatments showed the relationship between minimal water content observed during winter and maximal soluble sugars synthesized. Thus, the cold acclimation process appeared dependent on these physiological parameters (water and soluble sugar contents) through the interaction between air temperature and timing of leaf fall. (C) 2011 Elsevier BM. All rights reserved.