In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.