For the understanding of the relationship between protein structure and allergenicity, it is important to identify allergenic epitopes. Two methods to characterize primarily linear epitopes are compared using the major allergen from brown shrimp (Penaeus aztecus), Pen a 1, as an example. A recombinant peptide library was constructed and synthetic, overlapping peptides, spanning the entire Pen a 1 molecule, were synthesized and tested for specific IgE reactivity. Both methods identified IgE-binding of Pen a 1, however, the SPOTs procedure resulted in the identification of more epitopes of the major shrimp allergen Pen a 1 than the usage of the recombinant peptide library. For detection of specific IgE antibodies, the usage of 125I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies. The regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. If a given food allergen contains significant linear epitopes, which seems to be true for stable major allergens such as those of peanut and shrimp the SPOTs system may be more advantageous than the use of recombinant peptides libraries. However, if allergens are studied that contain more conformational epitopes, recombinant peptide libraries may help to identify the relevant epitopes. It has to be emphasized that no system for epitope identification will detect all epitopes and that the relevance of identified epitopes has to be confirmed with other methods such as inhibition studies, crystallographic analysis or the immunological evaluation of modified whole allergens.