Ole e 1 (NCBI entry gi|14424429) is the major allergen of Oleaceae family. Multiple isoforms and variants are present in varying degrees of distribution. In this report, we present a new approach to the resolution of multiple forms of Ole e 1 from whole antigen extracts, based on a preliminary chemical fractionation procedure followed by MALDI MS and MS/MS measurements. The characterization of Ole e 1 isoallergens was accomplished through the identification of the amino acid sequence including the glycosylation site and the structure of the glycan moieties. The structure feature of the identified Ole e 1.0102 (gi|2465127), main olive allergen [Olea europaea] (gi|13195753), Major pollen allergen Ole e 1 (gi|33329740) and Ole e 1c (gi|1362131) is represented by the point mutation K(106) --> I and by the presence of a glycan moiety. Two other variants Major pollen allergen (Allergen Ole e1) (Ole e I) (gi|14424429) and Ole e 1.0103 protein [Olea europea] (gi|2465129) were identified as nonglycosylated species. These results, partially in disagreement with Swiss-Prot annotation, were validated by matching the MALDI MS/MS spectra of the natural tryptic mixture with those obtained after deglycosylation.