BACKGROUND Milk hydrolysates, although frequently used as substitutes in cases of cow's milk allergy, show a reduced but never a complete abolishment of antigenicity and allergenicity. OBJECTIVE Our purpose was to determine the lower molecular weight limit of peptides to elicit skin reactions and to bind IgE antibodies in vitro. METHODS Using FPLC, an ultrafiltrated whey hydrolysate, was fractionated in different molecular weight fractions. Skin-prick tests were performed with the hydrolysate and its fractions in five cow's milk allergic children, and RAST inhibition tests were done using the serum of these children. RESULTS On the basis of the lowest extinction values between two peaks of the chromatogram, seven fractions with molecular weights between 15000 and 125 Da were obtained. Peptides of > 2600 Da elicited a clearly positive skin reaction and inhibited IgE-binding, while peptides of < 1400 Da did not give any positive skin reaction but were still able to inhibit to a small extent IgE-binding to the hydrolysate. CONCLUSIONS Our findings suggest that for skin reactivity peptides of > 1400 Da are needed. The minimal molecular mass for IgE binding in vitro appears to be situated between 1400 and 970 Da. Such peptides might be used to develop a safe formula for patients reacting to milk hydrolysates or even for tolerance induction.