A new approach is proposed to characterize polyampholytic gelatin hydrogels, cross-linked covalently with glutaraldehyde. An experimental methodology involving simple mechanical extension and compression and equilibrium swelling tests is developed to estimate relevant microstructural parameters and electrokinetic properties of this type of hydrogel, for different cross-linker to gelatin mass ratios. The polyampholytic cross-linked matrices are studied here in the framework of the rubber elasticity and thermodynamic swelling theories. The main purposes of this work are the estimations of average mesh size and toughness of the swollen hydrogels, and the determination of the feasibility of polyion complexation between cross-linked gelatin chains and bioactive macromolecules to be delivered through hydrogel biodegradation. (C) 2009 Elsevier Ltd. All rights reserved.