Dietary non-digestible carbohydrates reduce the development of cows' milk allergy in mice. In the present study, the contribution of CD25 þ regulatory T-cells (T reg ) was investigated using in vivo T reg depletion and adoptive transfer studies. Mice were orally sensitised with casein and fed a diet containing 2 % short-chain galacto-, long-chain fructo-and acidic oligosaccharides (GFA) or a control diet. Donor splenocytes of mice sensitised with casein and fed the GFA or control diet were adoptively transferred to naive recipient mice, which were casein-or sham-sensitised and fed the control diet. In addition, in vivo or ex vivo CD25 þ T reg depletion was performed using anti-CD25 (PC61). The acute allergic skin response upon intradermal casein challenge and casein-specific Ig were determined. Furthermore, T-helper (T H ) 1 and T H 2 cell numbers were analysed in the mesenteric lymph nodes. The oligosaccharide diet strongly reduced the development of the acute allergic skin response, which was abrogated by the in vivo anti-CD25 treatment. The diet enhanced the percentage of T H 1 cells and tended to reduce the percentage of T H 2 cells in casein-sensitised mice. Recipient mice were protected against the development of an acute allergic skin response when transferred with splenocytes from casein-sensitised GFA-fed donor mice before sensitisation. Ex vivo depletion of CD25 þ T reg abrogated this transfer of tolerance. Splenocytes from sham-sensitised GFA-fed donor mice did not suppress the allergic response in recipient mice. In conclusion, CD25 þ T reg contribute to the suppression of the allergic effector response in casein-sensitised mice induced by dietary intervention with non-digestible carbohydrates.Key words: Cows' milk allergy: Prebiotics: Non-digestible carbohydrates: Oligosaccharides: Regulatory T-cells: CaseinOne of the first manifestations of atopic disease is the development of cows' milk allergy (CMA) in young infants. Casein, in particular aS1-casein, is the main allergenic protein in cows' milk (1,2) . Non-digestible carbohydrates such as short-chain galacto-oligosaccharides (scGOS) and long-chain fructooligosaccharides (lcFOS) mimic structural and functional properties of neutral oligosaccharides such as present in human breast milk and are added to clinical nutrition and infant milk formulas since they may have health and immunepromoting capacities (3,4) . Previously, in a mouse model of orally induced CMA this 9:1 scGOS/lcFOS mixture (Immunofortisw; Nutricia, Zoetermeer, The Netherlands) was found to reduce the allergic effector response to whey protein (5) .Furthermore, in a clinical study with infants at high risk of developing allergic disease, the same carbohydrate mixture reduced the incidence of atopic dermatitis significantly (6) .Acidic oligosaccharides derived from pectin (pAOS) are non-digestible carbohydrates that mimic properties of human milk acidic oligosaccharides (7) . The combined use of neutral and acidic oligosaccharides was most effective in supporting the vaccinati...