Peanut allergy is one of the most severe food allergies. One effort to alleviate this problem is to identify peanut germplasm with lower levels of allergens which could be used in conventional breeding to produce a less allergenic peanut cultivar. In this study, we identified one peanut line, GT-C9, lacking several seed proteins, which were identified as Ara h 3 isoforms by peptide sequencing and named iso-Ara h 3. Total seed proteins were analyzed by one-dimensional (SDS-PAGE) and two-dimensional gel electrophoreses (2-D PAGE). The total protein extracts were also tested for levels of protein-bound end products or adducts such as advanced glycation end products (AGE) and N-(carboxymethyl) lysine (CML), and IgE binding. Peanut genotypes of GT-C9 and GT-C20 exhibited significantly lower levels of AGE adducts and of IgE binding. This potential peanut allergen iso-Ara h 3 was confirmed by peptide sequences and Western blot analysis using specific anti-Ara h 1, Ara h 2, and Ara h 3 antibodies. A full-length sequence of iso-ara h 3 (GenBank number DQ855115) was obtained. The deduced amino acid sequence iso-Ara h 3 (ABI17154) has the first three of four IgE-binding epitopes of Ara h 3. Anti-Ara h 3 antibodies reacted with two groups of protein peptides, one with strong reactions and another with weak reactions. These peptide spots with weak reaction on 2-D PAGE to anti-Ara h 3 antibodies are subunits or isoallergens of this potential peanut allergen iso-Ara h 3. A recent study suggested that Ara h 3 basic subunits may be more significant allergenicity than the acidic subunits.