Extracts from birch (Betula verrucosa) pollen were prepared at different pH, with constant pH monitoring and adjustment to preset values in the range 5.5-8.5. The total protein content of these extracts was directly correlated with the pH. Coomassie brilliant blue-stained isoelectric focusing and SDS-PAGE gels and immunoblot analysis demonstrated qualitative differences: some proteins were lost while others appeared when pH was changed. At pH 8.5, formerly unknown birch pollen allergens were detected with pI 9, 9.10, and 9.30 by about 30% of birch pollen-sensitive sera. Birch pollen extracts prepared at a pH close to neutrality, namely, 6.5 and 7.5, showed the greatest protein and different allergen diversity. Thus, extraction pH values are necessary to analyze the whole pattern of allergenic components in an extract.