Five distinct proteins with allergenic activity have been isolated from short ragweed pollen. We initially tested three of these, AgE, AgK, and Ra3, for reactivity with IgE antibodies by leukocyte histamine release and by the radioallergosorbent test (RAST). We found highly significant correlations between the reactivities of these allergens by leukocyte histamine release and by the RAST, consistent with the view that both procedures detected comparable allergenic activity. We next tested the allergenic cross-reactivity of all five ragweed allergens. AgE, AgK, Ra3, Ra4, and Ra5, by RAST inhibition. With solid-phase AgE the only nonhomologous inhibitor was AgK, which cross-reacted weakly and required a 140-fold mass excess of AgK compared to AgE. With solid-phase AgK both AgK and AgE produced significant inhibition; AgE was slightly more potent than the homologous AgK, Ra3 and Ra5 were allergenically unique, because only the homologous allergen produced 50% inhibition. Ra4 was weakly inhibited by AgE, Ra3, and Ra5 when these allergens were added in 300- to 5---fold mass excesses; this weak inhibition may represent either cross-reaction or cross-contamination. We found that RAST inhibition could be used as an assay for the individual ragweed allergens and we demonstrated the presence of all of the allergens in a whole ragweed extract. The sensitivity of the RAST inhibition assay ranged from 10 ng to 100 ng for 50% inhibition. Finally, the solid-phase ragweed allergens were used to determine the frequency of elevated IgE antibody levels in 65 patients with ragweed hay fever. Virtually all of the patients reacted with AgE (97%), while 88% reacted with AgK, 51% reacted with Ra3, 28% reacted with Ra4, and 17% reacted with Ra5. These results highlight the usefulness of the RAST as a specific and sensitive tool for immunochemical studies of allergens.