BACKGROUND Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species. Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens. OBJECTIVE In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens. METHODS Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunoelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE- inhibition studies. RESULTS Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunoelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients' IgE. CONCLUSIONS Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy.