A number of environmental cues including short day photoperiod (SD) and low temperature (LT) are known to interact in triggering growth cessation, cold acclimation and other adaptive responses in temperate-zone tree species. Proper timing of these responses is particularly important for survival of trees in the boreal and subarctic regions. Therefore, we used a northern tree species, silver birch (Betula pendula Roth) as an experimental model to investigate the effect of SD and LT on development of freezing tolerance and on levels of endogenous abscisic acid (ABA) in short-term experiments under controlled conditions. We characterized differences in SD and LT-induced cold acclimation between three different climatic ecotypes from southern, central and northern habitats. The results demonstrated that cold acclimation was rapidly triggered by exposing the plants to SD or LT, and that a combination of the different treatments had an additive effect on freezing tolerance. Freezing tolerance induction was not uniform in the different tissues, the buds and leaves developed freezing tolerance more rapidly than the stem, and the young leaves had a higher freezing tolerance than the old leaves. The ability of the leaves to respond to SD and LT and similarity of the bud and leaf responses indicate that birch leaves provide a rapid and convenient system for studies on molecular mechanisms of cold acclimation. Development of freezing tolerance was dependent on the climatic ecotype, the northern ecotype was clearly more responsive to both SD and LT than the two more southern ecotypes. Development of freezing tolerance induced by SD and LT was accompanied by transient changes in ABA levels. These alterations in ABA levels were ecotype-dependent, the northern ecotype reacting more strongly to the environmental cues.