Diffusion coefficients of sodium chloride, sodium nitrate and oxygen were determined for heterotrophic biofilms. The biofilms were cultivated under different hydrodynamic and substrate loading conditions in tubular reactors resulting in biofilm densities between 3 and 40kg/m(3) dry mass. Quantifying solute diffusion in the biofilm for these biofilms allowed to specifically evaluate the influence of biofilm density on diffusion while keeping other factors such as the type of substrate, the inoculum, and the reactor type constant. Two methods were used to measure diffusion coefficients. The two-chamber method was used to quantify the diffusion of sodium chloride and sodium nitrate. The diffusion coefficients for oxygen were measured based on oxygen concentration profiles in the biofilm measured using microelectrodes. The ratio between the diffusion coefficient in biofilm and water (f(D) = D-F/D-W) was found to be lower than 1 in the majority of experiments. A clear correlation between f(D) and biofilm density was found where f(D) decreased with increasing biofilm density. For mean biofilm densities in the range of 10-20kg/m(3) f(D) can be approximated between 0.5 and 1. For larger densities of 20 or 35kg/m(3) f(D) can be approximated as 0.8 or 0.4, respectively. For densities higher than 35 kg/m(3) f(D) is below 0.6. Advective transport mechanisms that would have resulted in f(D) > 1 can be neglected in the biofilms cultivated. (c) 2005 Elsevier Ltd. All rights reserved.