The present experiments were designed to evaluate the effective thickness of the unstirred layers in series with native and porous (i.e., in the presence of amphotericin B) lipid bilayer membranes and, concomitantly, the respective contributions of membranes and unstirred layers to the observed resistances to the diffusion of water and nonelectrolytes between aqueous phases. The method depended on measuring the tracer permeability coefficients for the diffusion of water and nonelectrolytes (P(DDi), cm sec(-1)) when the aqueous phase viscosity (eta) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (alpha(t), cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (P(mmi), cm sec(-1)) were computed from, respectively, the slope and intercept of the linear regression of 1/P(DDi) on eta. In both the native and porous membranes, alpha(t) was approximately 110 x 10(-4) cm. The ratio of P(f), the osmotic water permeability coefficient (cm sec(-1)) to P(mmH2O) was 1.22 in the native membranes and 3.75 in the porous membranes. For the latter, the effective pore radius, computed from Poiseuille's law, was approximately 5.6 A. A comparison of P(mmi) and P(DDi), indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H(2)O, urea, and glycerol, while the remainder was referable to the unstirred layers.