Two chromatographic forms (A and B) of the rapidly released short ragweed (Ambrosia elatior) pollen allergen Ra6 were isolated to antigenic ultrapurity by a combination of membrane filtration and ion exchange and gel filtration chromatography. Both Ra6 forms had a m.w. of 11,500 by gel filtration chromatography and 8000 by SDS-PAGE, and each consisted of two cathodically migrating bands on agarose electrophoresis at pH 8.5. Amino-terminal amino acid sequencing analysis detected two similar sequences (sequence 1 and sequence 2), which were present in different proportions in each of the chromatographic forms of Ra6. Among a ragweed-positive population sample (n = 101), 21% had specific IgE antibody (ab) and 42% specific IgG ab toward Ra6A; among ragweed-negative subjects (n = 260), 10.8% had IgG ab. We were unable to discriminate between Ra6A and Ra6B immunologically with the use of hyperimmune animal antisera or IgE and IgG ab from five Ra6-allergic humans. It appears that Ra6 is composed of at least four closely related isoallergens of similar size but slightly different charges and amino acid sequences, which appeared to be antigenically and allergenically indistinguishable according to present evidence.