Mass transfer limitations severely impede the performance of bioreactions involving large molecules by gel-entrapped microorganisms. This paper describes a quantitative investigation of such diffusional limitations in agar gel membranes. Sucrose and commercial dextran fractions with (weight-average) molecular weights ranging from 10,000 to 2,000,000 Da were used as standard diffusants. For all tested solutes but sucrose, the values of the agar/water partition coefficients highlighted steric hindrance at the entrance of the membrane pores. The effective diffusivity of sucrose in agar was similar to that in water. All dextran fractions, however, displayed restricted diffusion in the agar membranes. Their effective diffusivities were a decreasing function of the agar content of the gel membrane (0.5, 1.0, or 1.5% w/v). The effective diffusivity in a given membrane decreased as the molecular weight of the diffusing molecule increased. T500 (M(w)BAR = 470,000 Da) and T2000 (M(w)BAR = 1,950,000 Da) fractions were unable to diffuse through 1.0 or 1.5% agar membranes. The diffusion data did not agree with the classical (Renkin) model for a hard sphere diffusing through a cylindrical pore. These results are discussed in terms of gel and diffusant characteristics.