This study investigated the quantity and distribution of extracellular polymeric substances (EPS) in aerobic granules. Results showed that EPS play an important role in the formation and stabilisation of granules. The content of EPS significantly increases during the first weeks of biogranulation. An analysis of EPS in the granules revealed that the protein level was 5 times higher than in polysaccharides. The increase of protein content correlated with the growth of cell hydrophobicity (r2 = 0.95). EPS and hydrophobicity are important factors in cell adhesion and formation of granules. The aim of this work was also to determine the distribution of EPS in the granule structure. In situ EPS staining showed that EPS are located mostly in the center of granules and in the subsurface layer. The major components of the EPE matrix are proteins, nucleic acids and β-polysaccharides. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on protein content.