As part of a study to probe the immunochemical basis for allergenic cross-reactivity among grass pollens, a series of useful reagents has been prepared. The major grass-pollen allergen, designated group I (GpI), was isolated from five grass pollens (meadow fescue, June grass, sweet vernal grass, redtop grass, and perennial ryegrass). The purified GpI antigens were used to immunize individual groups of BALB/c mice. A total of 123 hybridoma-derived anti-GpI monoclonal antibodies (mAbs) was produced. These mAbs were used to evaluate the antigenic relationship among the GpI antigens by means of two types of ELISA. The experiments revealed a high level of epitope diversity and demonstrated a wide range of antibody specificities. A cross-reactivity ELISA was used to identify and compare antigenic determinants on the GpI molecules, and it was possible to define mAbs with specificities unique for the immunizing allergen and other mAbs that cross-reacted with one or more other members of the test panel of allergens. These murine mAbs reflect the range of specificities present in sera from grass-allergic individuals.