Abstract The importance of apple allergens, in particular Mal d 1, a Bet v 1 homologue for the pollen-fruit syndrome in Northern Europe, and Mal d 3, responsible for true fruit allergy in Southern Europe, has been repeatedly emphasized. However, little is known about the distribution pattern of major allergens in fruits and whether differences exist among different cultivars. Transcript expression of Mal d 1 isoforms and Mal d 3 was examined by RealTime-PCR and Northern analysis, respectively. An immuno-tissue-print (ITP) assay was developed to localise major allergens in apple fruit tissue and a Mal d 1 specific, patient independent ELISA was established. ITP analyses show that Mal d 1 and Mal d 2 are distributed throughout the apple pulp and peel, while Mal d 3 is restricted to the peel. Data obtained by ELISA reveal a variation of Mal d 1 content ranging from 0.84 to 33.17 μg/g fresh weight in 39 selected cultivars. Different apple cultivars show a markedly different expression of major allergens; this finding will influence the development of diagnostic tools as well as the dietary management of allergic individuals.