In the present work, porous gelatin scaffolds were prepared by cryogenic treatment of a chemically cross-linked gelatin hydrogel, followed by removal of the ice crystals formed through lyophilization. This technique often leads to porous gels with a less porous skin. A simple method has been developed to solve this problem. The present study demonstrates that the hydrogel pore size decreased with an increasing gelatin concentration and with an increasing cooling rate of the gelatin hydrogel. Variation of the cryogenic parameters applied also enabled us to develop scaffolds with different pore morphologies (spherical versus transversal channel-like pores). In our opinion, this is the first paper in which temperature gradients during controlled cryogenic treatment were applied to induce a pore size gradient in gelatin hydrogels. With a newly designed cryo-unit, temperature gradients of 10 and 30 degrees C were implemented during the freezing step, resulting in scaffolds with average pore diameters of, respectively, +/- 116 and +/- 330 mu m. In both cases, the porosity and pore size decreased gradually through the scaffolds. Pore size and structure analysis of the matrices was accomplished through a combination of microcomputed tomography using different software packages (mu CTanalySIS and Octopus), scanning electron microscopy analysis, and helium pycnometry.