Technical Abstract: Ara h 1, a vicilin, Ara h 2, a 2S albumin, and Ara h 3, a legumin, are major33 peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but co-sensitization to all three allergens is correlated with the severity of patients’ symptoms. We investigated whether co-sensitization to these three allergens is caused by IgE cross-reactivity, despite the fact that they do not display obvious structural or sequence similarities. IgE cross-inhibitions were performed using purified Ara h 1, Ara h 2, and Ara h 3, and sera from 10 peanut-allergic subjects. Following an in silico search for similar peptides, IgE binding inhibition and basophil activation assays, using synthetic peptides were performed. Ara h 1 completely inhibited IgE binding to Ara h 2 (median 96%) and Ara h 3 (median 100%), whereas, IgE binding to Ara h 1 and Ara h 3, was partially inhibited by Ara h 2 (medians 81% and 73%). IgE binding affinity for Ara h 2 was greater than for Ara h 1 and Ara h 3. A comparison of sequences showed that these non-homologous peanut allergens contain several similar surface exposed peptides. IgE binding to Ara h 2-derived peptides was completely inhibited by Ara h 1 and Ara h 3. A mixture of these peptides reduced the capacity of the three allergens to induce mediator release (12-49%). Occurrence of similar sequences in the three major peanut allergens account for the high extent of cross-reactivity among them.