In a methanogenic biofilm reactor, fatty acids diffused in the symbiotic biofilms and were degraded to methane and carbon dioxide. The diffusion of fatty acids was retarded due to the presence of gelatinous biomass and the formation of tiny gas bubbles within the biofilms. The effective diffusivities of fatty acids (acetate, propionate and butyrate at pH 7.1) were measured 'in situ' in active methanogenic biofilms after the fatty acid concentration profiles approached the steady state in the biofilms. By solving reaction-diffusion models for each acid after the three boundary conditions had been experimentally determined with a specially designed diffusion-cell, the effective diffusivities of acetate, propionate and butyrate in methanogenic biofilms at 35-degrees-C were found to be 0.54, 0.60 and 0.36 x 10(5) cm2/s, respectively. The diffusion coefficients in biofilms were 33% of those in water solution at infinite dilution. Compared with the diffusion in water, the effective diffusivities in biofilms were reduced by about 54% due to the presence of solid biomass, and a further retardation in diffusion was attributed to the formation of tiny gas bubbles (CH4 and CO2) in the active methanogenic biofilms.