Biofouling is a major impediment in reverse osmosis (RO) and nanofiltration (NF) cross-flow processes. Due to the cross-flow component, feedwater microorganisms are transported to the membrane surface where they adsorb, forming a fouling layer. Once attached, the microorganisms grow and multiply at the expense of feedwater nutrients, forming a biofouling layer which reduces permeate fluxes and damages the membrane. Prefiltered secondary effluents and synthetic media simulating secondary effluents were applied for studying NF membrane biofouling, using 120 cm(2) area-polyamide tubular membranes of 200 Da MWCO. After operation the membranes were dismounted and segmented in slices which were analyzed by spectroscopic, microscopic and molecular techniques. Fully developed biofilm was found on the membranes and biofouling appears to be the predominant form of fouling composed of bacteria and extracellular polymers, while the size and composition of biofilm, population seem to be convergent to finite number. Microbial populations identified by sequence homology corresponded phylogenetically to Flavobacterium sp, Pseudomonas, Ralstonia and Cytophaga regardless of the conditions applied. Microscopic observations revealed presence of protozoa as well. Bacterial counts in biofilm accounted for approx. 10(7) CFU cm(-2) regardless of the feed applied. Although biofouling layer over the membrane reduced flux, biofouling development improved brine rejection (50-70%). FTIR spectra of the organic matter accumulated on the NF membrane showed the presence of proteins and amino acids and a high level of aliphatic and polysaccharide residues.