The effects of epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) on the healing process of tympanic membrane perforations were studied in guinea pigs. The diameters of the perforations were measured daily using an operating microscope. The perforations healed rapidly, when hypertrophy of the epithelial layer was accelerated by EGF. Closure of the perforations was inhibited, when regeneration of the middle layer was disturbed by TGF-β. Regression analysis on the diameters of the perforations revealed that spontaneous healing of the perforations was accelerated during the second half of healing process with simultaneous hypertrophy of the middle layer.