The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage. © 2012 Pleiades Publishing, Ltd.