Hydrogel membranes were prepared by radiation and chemical cross-linking of poly(vinyl alcohol) (PVA) in aqueous solutions. Effects of PVA concentration, PVA molecular weight and radiation dose, as well as concentration of cross-linking agent, in the case of the chemical cross-linking procedure, on the permeation of insulin, albumin and immunoglobulin (IgG) through the membranes were investigated. Glucose permeation was also studied. The cross-linking density affected the size of the macromolecular mesh of the hydrogel network and thus the water content of the membrane responsible for the diffusion of the solutes. The diffusion coefficient linearly increased for all the solutes with increasing water content in the PVA hydrogels, indicating that diffusion occurs primarily through the water hydrating the polymer network. The permeability study showed that the water content as well as the mesh size had an influence on the diffusion of low molecular weight glucose and insulin. Although the diffusion of higher molecular weight solutes, such as albumin and IgG, was not so much affected by the mesh size of elaborated PVA hydrogel membranes, the diffusion of these proteins was very low.