Immune reactivity is a key issue in the evaluation of the quality of recombinant allergens as potential reference materials. Within the frame of the CREATE project, the immune reactivity of the natural and recombinant versions of the major allergens of birch pollen (Bet v 1), grass pollen (Phl p 1 and 5), olive pollen (Ole e 1), and house dust mite (Der p 1 and 2, and Der f 1 and 2) was analysed. The IgE binding capacity of the allergens was studied by direct RAST and RAST inhibition, and their biological activity by basophil histamine release, using sera of allergic patients selected across Europe. For birch pollen, rBet v 1 is an excellent mimic of the natural allergen. For grass pollen, rPhl p 1 showed a significant lower IgE reactivity and was not considered a suitable candidate, whereas rPhl p 5a exhibited an immune reactivity closer to that of its natural counterpart. For olive, rOle e 1 had a lower IgE binding capacity in RAST but a higher biological activity in histamine release. For house dust mite, recombinant group 1 allergens were significantly less potent than their natural counterparts, but recombinant group 2 allergens were close mimics of their natural homologues.