Rationale: Protein unfolding reduces significantly the allergenic activity of the most potent peanut allergen Ara h 2. This allergen also contains major sequential IgE-binding epitopes, especially in a flexible region comprising the repetitive post-translationally modified peptide DPYSPOHS, with a hydroxyproline (HYP). We then investigated the relative contributions of sequential and conformational epitopes to the allergenic activity of Ara h 2. Methods: Peptides overlapping the HYP-domain were synthesized with or without the prolines hydroxylated. A variant lacking the HYP-domain was generated by deletion mutagenesis. IgE reactivity of 19 peanut-allergic patients toward synthetic peptides and recombinant Ara h 2 variants was determined by IgE-binding inhibition assays and by degranulation tests of rat basophil leukemia (RBL) SX38 cells sensitized with patients’ IgE antibodies. Results: The peptide encompassing the longest HYP-domain exhibited an inhibitory capacity similar to that of the full-length unfolded Ara h 2, but only when prolines were hydroxylated. Deletion of the HYP-domain did not prevent the variant to refold correctly and to display Ara h 2 conformational epitopes. The IgE-binding capacity of Ara h 2 was then recapitulated with an equimolar mixture of the deletion variant and the synthetic HYP-peptide. Surprisingly, the HYP-peptide was also able to trigger the degranulation of RBL cells as efficiently as the unfolded Ara h 2, which retained an allergenic activity with most of the tested sera. Conclusions: Proline hydroxylation is critical for Ara h 2 allergenicity and peptides overlapping the HYP-domain, as short as 15 and 27 amino acid residues, retain an anaphylactic potential.