The structure of fouling layer determines the pressure drop across the fouling layer. Three-dimensional distributions of nucleic acids, proteins, alpha-D-glueopyranose polysaccharides, beta-D-glucopyranose polysaccharides and lipids in the biofouling layer that is formed on a mixed cellulose ester membrane were determined using a six-fold staining protocol combined with confocal laser scanning microscopy (CLSM). Based on the three-dimensional volumetric grid model of the fouling layer structure observed from the series of CLSM images, the intra-layer flow field during filtration was simulated using commercial software. The effective permeability of the fouling layer was estimated to be 2.65 x 10(-12) m(2), which determines the upper estimate on the permeability of the fouling layer. The pores were categorized according to their diameters, using the maximum convex perimeter approach, and then the effects of the blocking pores on the permeability of the fouling layer were investigated. Blocking the large pores that accounted for 15% of the porosity reduced the mean permeability by 58%. (c) 2006 Elsevier B.V. All rights reserved.