Pru p 3, a member of the lipid transfer protein family, is considered a major allergen from peach as it often induces serious allergic reactions in peach-allergic individuals. The high resistance of Pru p 3 to processing treatments and to digestion or enzymatic hydrolysis is probably the cause of the severity of this fruit allergy. The aim of this study was to determine the effect of treatment with a large number of proteases from different origins (vegetal, animal and microbial) on the degradation and allergenicity of Pru p 3. To perform this study, Pru p 3 was previously isolated using cation exchange chromatography and ultrafiltration, and the purified protein was incubated with proteases under different conditions. The results showed that only two of the fifteen proteases assayed were able to efficiently degrade the protein at acidic pH, as determined by SDS-PAGE. These two commercial acid proteases, derived from Aspergillus niger, decreased by more than 95% the immunoreactivity of Pru p 3 by ELISA using specific rabbit IgG, giving peptides lower than 3.2 kDa as determined by MALDI-TOF mass spectrometry. The hydrolysates obtained showed a greater than 70% decrease in reactivity of IgE compared to untreated Pru p 3 using three pools of sera from peach allergic individuals. Furthermore, when hydrolysates were tested by the prick test, in more than 90% of peach-allergic patients the average size of the wheal significantly decreased by between 72% and 85%. The results suggest that the acid protease from Aspergillus niger could be used to obtain novel hypoallergenic products more tolerable for peach-sensitive individuals.