AbstractThe recent availability of the honey‐bee Apis mellifera genome and trascriptome of both the female castes, has stimulated new efforts in investigating the protein composition of royal jelly (RJ), its role in caste differentiation and its quality and typicality by a proteomic approach. This study is aimed both to separate and identify proteins of royal jelly and to detect some of them in honey‐bee pollen‐bread by using two‐dimensional gel electrophoresis, mass spectrometry and by de novo sequencing. All the identified proteins belonged to the Apis mellifera genome. Apalbumin 1 was also confirmed to be present in honey‐bee pollen‐bread where the presence of apalbumin 2 was also found. In addition several fragments of apalbumin 1 and apalbumin 3 were also found in RJ. These could be the result of protease activity other than that of serine‐protease. This study is a contribution to the description of royal jelly proteome.