The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pi values of 5.2 7.5 and 4.8 5 9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens u combination of enzymatic cleavage of electrophoretically separated proteins and iminunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels. proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.