Although extract of velvet grass pollen is an important ingredient of commercial allergen extracts for immunotherapy, it is not defined concerning its major allergenic components. We have investigated the extract of velvet grass pollen by Western blotting using the sera of 97 patients and identified two major allergens of 34 and 30 kD. By two-dimensional immunoblotting with monoclonal antibodies and lectins, and by biochemical analysis the 34-kD protein was identified as a group I grass pollen allergen and the 30-kD protein as a group V grass pollen allergen. According to the nomenclature for grass pollen allergens we designated these allergens Hol l 1 and Hol l 5, respectively. Fragmentation of the velvet grass pollen extract by CNBr provided the first information on the localization of IgE binding epitopes on these two allergens. With Hol l 1 and Hol l 5 we identified two important components of commercial allergen extracts.