The diffusion coefficient of glucose was measured in biological flocs under various growth conditions. Significant differences were observed with different carbon sources and carbon to nitrogen ratios. The diffusion coefficients were calculated from direct measurements of mass flux through a series of membrane filters separated by a metal template which contained the floe. The template assembly was prepared by packing the floe material into the template and placing a .45|j, membrane filter on either side of the template. The assembly was then placed in a modified plastic Millipore filter apparatus having the lower portion filled with a glucose solution (1 mg/1) and the top section containing deionized water. Increasing glucose concentration in the top was related to mass flux through the floe by measuring the flux through the membrane filters separately. Measurements were replicable within 8%. A 2^3 factorial analysis, using carbon source (glucose or methanol), carbon to nitrogen ratio (5:1 or 5:1), and temperature (2°C or 3°C) as independent variables, indicated significant effects at the 99% level for all three variables.