Two isoallergenic forms of short ragweed (Ambrosia elatior) allergen Ra5 were isolated in ultrapure form by a combination of molecular sieving (through Amicon hollow-fiber cartridges), cation-exchange chromatography, and gel filtration. The two forms, Ra5A and Ra5B, exhibited charge differences by CM-Sephadex chromatography and agarose electrophoresis at pH 8.5. The A form as identical to conventional Ra5 by its physical and chemical properties and was more cationic than the previously undescribed B form. Ra5A and Ra5B had identical m.w. of 5000 by SDS-PAGE. Their amino-terminal sequences of 30 residues showed no differences from each other or from the previously published sequence of the major form of Ra5. The two forms were antigenically indistinguishable when using hyperimmunized animal antisera to Ra5 and crude ragweed extract. Although the IgG and IgE ab of most Ra5-responder patients could not discriminate between the two Ra5 forms, significant small differences in binding to Ra5A and Ra5B were consistently observed in 10 to 15% of the patients studied. Further analysis of the Ra5A preparation suggested a purity of at least 99.5% and probably greater than or equal to 99.9%.