Hydrogel membranes with mesh size asymmetry, created by cross-linking poly(vinyl alcohol) (PVA) under a glutaraldehyde gradient, were characterized with laser scanning confocal fluorescence microscopy. A fluorescent label, 5-{[4,6-dichlorotriazin-2-yl] amino}-fluorescein (DTAF) was attached to PVA, which then was used to prepare homogeneous and asymmetric hydrogel membranes. Using homogeneous membranes of uniform mesh size, fluorescence intensity was correlated to water content to characterize the mesh-size asymmetric hydrogels. Structural asymmetry was clearly present in the gradient-modified membranes from the intensity as a function of membrane depth. Average water contents were calculated and compared to measured values. The permeabilities of creatinine, Fab and IgG for the asymmetric membranes were predicted from experimental permeability data as a function of water content for homogeneous membranes, assuming a sum-of-resistances model. The calculated water contents and predicted solute permeabilities compared well to the experimental values.