The kinetic formation of biofilms developing on nanofiltration (NF) membranes was studied for 2 years in the water production unit of Méry-sur-Oise, France. New membranes were set up in a pilot train integrated to the plant and autopsied after operation for 7, 80, 475 and 717 days. The biofouling layer was studied by confocal laser scanning microscope after 4',6-diamidino-2-phenyindole dihydrochloride and lectin staining, and by attenuated total reflectance-Fourier transform infrared spectroscopy and rheology experiments. Three stages of biofilm growth were discriminated: (1) the presence of sessile microcolonies embedded in an exopolymeric matrix (after filtration for seven days); (2) membrane coverage expansion through microcolony development and biofilm growth in three dimensions (up to 80 days filtration); and (3) biofilm maturation by densification (after filtration for 80-717 days). Biofilm maturation resulted in total coverage of the membrane surface and matrix residue diversification, development of the polysaccharide network, and the strengthening of matrix cohesion through viscosity and elasticity increases. The wettability and permeability of the fouled NF membranes decreased quickly and continuously throughout the biofilm development process. The longitudinal pressure drop (LPD) increased only after the biofilm reached a quantitative threshold. The decline in membrane permeability may be the result of contributions from many fouling mechanisms but the LPD was more substantially influenced by biofilm development.