We have purified a 24/25 kd allergen from orchard grass pollen (Dactylis glomerata) that has an allergenic potency similar to that of the major group I allergen. We provisionally named this allergen grass 4B1 after the monoclonal antibody used for its identification and purification. This monoclonal antibody was obtained by immunizing mice with whole Lolium perenne-pollen extract and by screening the antibody producing hybrids for reactivity with Dactylis glomerata-pollen extract. Grass 4B1 is physicochemically separable from the group I allergen. Polyclonal rabbit antibodies to grass 4B1 do not react with group I allergen or vice versa. Ninety-five sera with IgE antibodies to grass pollen were tested for IgE antibodies to grass 4B1, and greater than 90% was positive in this test. The median response to grass 4B1 was 70% of that to Lol p I.