In case of bulk loss of tissue or non-healing wounds such as burns, trauma, diabetic, decubitus and venous stasis ulcers, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate the cell proliferation and stimulate the healing process. In this study, synthesis of a novel polymeric bilayer wound dressing containing epidermal growth-factor (EGF) -loaded microspheres was aimed. For this purpose, a natural, nontoxic and biocompatible material, gelatin, was chosen as the underlying layer and various porous matrices in sponge form were prepared from gelatin by freeze-drying technique. As the external layer, elastomeric polyurethane membranes were used. Two different doses of EGF was added into the prepared gelatin sponges (1 and 15 microg/cm2) to activate cell proliferation. EGF addition was carried out either in free form or within microspheres to achieve prolonged release of EGF for higher efficiency. The prepared systems were tested in in vivo experiments on full-thickness skin defects created on rabbits. At certain intervals, wound areas were measured and tissues from wound areas were biopsied and processed for histological examinations. The wound areas decreased upon low-dose EGF application but the difference between the affects of free EGF and microsphere loaded EGF was not so distinct. Upon increasing the dose of EGF by a factor of 15, it was observed that controlled release of EGF from microspheres provided a higher degree of reduction in the wound areas. Histological investigations showed that the prepared dressings were biocompatible and did not cause any mononuclear cell infiltration or foreign body reaction. The structure of the newly formed dermis was almost the same as that of the normal skin.