A multistep procedure has been developed and applied to extract and purify proteins from lemon fruit flavedo. 2DE, LC-ESI-MS/MS, and bioinformatics were used to detect the high abundance of the germin-like glycoprotein Cit s1, a powerful allergen in humans. Peptide alignments against Citrus EST repositories gave the best scores with the C. sinensis cDNA (gi|188354270/EY710037), annotated as unknown sweet orange fruit protein; additional BLAST of peptides against NCBI databases gave high sequence identities with sequence of orange Cit s1 (gi|52782810/P84159), suggesting that the unknown sweet orange fruit protein is consistent with the Cit s1 protein. Peptides of Cit s1 were detected in 17 spots ranging from 120 to 20 kDa, pointing out that in the flavedo of lemon the Cit s1 may be expressed as several isoforms of which the 120 kDa isoform is the largest monomer and the 20 kDa is the smallest one. This finding adds information about the features of Cit s1, because it has been previously reported as a unique monomeric glycoprotein of 24 kDa.