Background: The major allergen of birch pollen, Bet v 1, is present in structurally slightly different isoforms. It has been postulated that certain isoforms show a distinct ability to bind birch pollen–specific IgE, although the T-cell response remains similar. Objective: We verified the hypothesis of a distinct allergenicity but similar T-cell immunogenicity of 2 isoforms in birch pollen–allergic subjects by in vivo tests and an in vitro assay for T-cell stimulation. Methods: Forty-eight birch pollen–allergic, 11 grass pollen–allergic, and 10 nonatopic control individuals were tested with 10-fold increasing concentrations (0.01 to 10.0 μg/mL) of recombinant (r) Bet v 1a and rBet v 1d by skin prick test (SPT), intradermal test (IDT), and conjunctival provocation test (CPT). An allergen-specific proliferation assay was performed on 21 patients with the 2 recombinant and the natural birch pollen allergens. Results: In each test system only birch pollen–allergic subjects but no controls reacted to the recombinant allergens. A positive in vivo response to 10 μg/mL of rBet v 1a was observed in 21 of 48 by SPT, in 48 of 48 by IDT, and in 33 of 48 by CPT. In contrast, the IDT response to 10 μg/mL of rBet v 1d was reduced by a factor of 100 because it was equivalent to the response to 0.1 μg/mL of rBet v 1a. rBet v 1d failed to elicit a positive reaction in SPT and CPT. The proliferative response of T cells was similar for both recombinant isoforms because 8 of 21 individuals reacted to rBet v 1a and 6 of 21 to rBet v 1d. Only 1 subject had a positive reaction to rBet v 1d alone. Conclusion: The natural isoforms rBet v 1a and rBet v 1d differ in their ability to bind IgE but are similar in their immunogenicity for T cells. Thus rBet v 1d might be a promising candidate for use in immunotherapy of birch pollen–allergic individuals. (J Allergy Clin Immunol 1999;104:1239-43.)