A role of non-reducing sugars like sucrose and raffinose in the protection of plant cells against damage during freezing has been proposed for many species, but reports on physiological effects are conflicting. Non-aqueous fractionation of mesophyll cell compartments in Arabidopsis thaliana was used to show that sucrose and raffinose accumulate in plastids during low temperatures, pointing to a physiological role in protecting the photosynthetic apparatus. Comparing a previously described raffinose synthase (RS) mutant of A. thaliana with its corresponding wild type, accession Col-0, revealed that a lack of raffinose has no effect on electrolyte leakage from leaf cells after freeze-thaw cycles, supporting that raffinose is not essential for protecting the plasma membrane. However, in situ chlorophyll fluorescence showed that maximum quantum yield of PS II photochemistry (F (v)/F (m)) and other fluorescence parameters of cold acclimated leaves subjected to freeze-thaw cycles were significantly lower in the raffinose synthase mutant than in the corresponding wild type, indicating that raffinose is involved in stabilizing PS II of cold acclimated leaf cells against damage during freezing.