The major allergen of birch pollen BV45 (Bet v I) was previously isolated by molecular weight exclusion chromatography and eluted in the molecular weight region of 15-29 KD. Further purification of this fraction on an SP-Trisacryl M cation exchange matrix allowed 6 peaks of which which the 4th (BV4A4) and 6th (BV4A6) included two dominant IgE-binding birch pollen isoallergens designated Bet v I and Bet v II. Final purification, using the 'Applied Biosystems' Peptide Micro Separation System, revealed two sharp peaks with a high degree of homogeneity. This was ascertained by automatic N-terminal amino acid (AA) sequence analyses which showed high average repetitive yields of the phenyl-thiohydantoin (PTH) AAs of the isoallergens sequenced. N-terminal AA analyses of the two fractions allowed 51 cleavages with correct identifications of PTH AAs for 3 replicates. The sequence data of the two isoallergens showed large homologies with the hazel pollen allergen, Cor a I, the birch pollen allergen, Ag 23, and the translated cDNA sequence derived from cloning birch pollen allergen genes. The sequence homologies support that Betula verrucosa allergens were derived from a gene family expressing several isologous allergens, 2 of which with 13 variable residues in a segment of 51 AAs. The antigenicity of the two fractions, Bet v I and Bet v II, was demonstrated by fused rocket immunoelectrophoresis (FRIE) and by crossed immunoelectrophoresis (CIE) giving single symmetrical antigenic precipitation.(ABSTRACT TRUNCATED AT 250 WORDS)