Of the several latex proteins cloned and expressed, the rubber elongation factor, Hev b 1, and the closely related Hev b 3, represent two major allergens associated with latex allergy. Although both allergens demonstrated IgE binding with sera from latex allergic patients, it was not known whether these two molecules shared any epitopes. Hence, in the present study using health care workers (HCW) and spina bifida (SB) patients with latex allergy, we investigated the IgE binding epitopes in Hev b 1 and Hev b 3. Recombinant Hev b 1 and Hev b 3 were expressed in a prokaryotic expression system, while overlapping decapeptides of Hev b 1 and Hev b 3 were synthesized on derivatized cellulose membrane. Eight IgE binding epitopes for Hev b 1 and eleven for Hev b 3 were identified using sera from latex allergic patients with SB. On further analysis of synthetic peptides encompassing these epitopes, similar IgE antibody reactivity was demonstrated with three Hev b 1 epitopes b1E3, b1E5, b1E6 and two Hev b 3 epitopes; b3E10 and b3E 11. For Hev b 1, a unique IgE binding epitope was identified in the region of amino acid residues 16-25. In competitive ELISA, peptides bIE2 and bIE4 together inhibited 58% of IgE binding of Hev b 1, while b3E5 showed 22% inhibition in the IgE binding of Hev b 3. The results of the present study suggest that the understanding of linear and conformational IgE epitopes in the major latex allergens may provide better insight into the structure-function relationship of the allergens, and may lead to the development of better patient care and management strategies in latex allergy.