The microstructure of polyampholyte gelatin hydrogels crosslinked covalently with fully hydrolyzed poly(vinyl alcohol) is characterized. The experimental methodology involves simple mechanical extension, spectroscopic and thermal methods (FTIR, XRD, DSC, TGA) and equilibrium swelling test to quantify relevant microstructural parameters and electrokinetic properties of this polyampholyte-polypeptide hydrogel, for a well defined range of crosslinker to gelatin mass ratios. These polyampholyte crosslinked matrices having different positive effective electrical charge are studied within an experimental and theoretical framework of their elastic and swelling properties. Estimations of average mesh size and toughness of the swollen hydrogels are provided. The feasibility of polyion complexation between positive crosslinked gelatin chains and negative bioactive macromolecules to be delivered through hydrogel biodegradation is analyzed. Several additional hydrogel properties are estimated like shear modulus, average molecular mass of network strands, swelling ratio, Flory-Huggins interaction parameter, hydrogel electrostatic zeta-potential and average effective charge number per chain before and after the crosslinking process to quantify gelatin chain cationization. (C) 2013 Elsevier Ltd. All rights reserved.