To determine the distribution of epithelial stem cells on the tympanic membrane, the changes of stem cells after tympanic membrane perforation, the in vitro culture growth characteristics of different parts of the tympanic membrane epithelium, and to establish a method for in vitro culture and proliferation of tympanic stem cells. Four young SD rats and four adult SD rats were observed as normal tympanic membrane, and 28 adult rats were perforated at the central 2 mm of tympanic membrane tension. Frozen sections of the tympanic membranes were taken at different times after perforation for immunohistochemical detection of β1 integrin and keratin 19. 30 adult SD rats’ tympanic membranes were damaged with mitomycin C on the mucosal surface and divided into two parts, the tympanic ring epithelium and the tense central epithelium, to compare the cell proliferation time of cells in both the tympanic ring and the tense part. The cells of the tympanic ring were classified according to the apposition time using 1 hour as the standard. Keratin 19 and β1 integrin-positive cells were distributed in the tympanic ring and hammer bone stalk region in the tense part and scattered in the relaxed part, with no positive cells in the central part of the tense part. There was no difference between juvenile and adult rats. The number of positive cells in the tympanic ring and hammer bone stalk increased after acute perforation of the tense part, and there was no positive staining at the edge of the perforation. In vitro culture of tympanic ring epithelial cells took significantly less time to multiply than the central epithelium of the tense end, and cells that adhered to the wall within 1 hour were actively growing, produced larger colonies, and were rich in epithelial stem cells. Epithelial stem cells were distributed in the tympanic ring and hammer bone stalk regions in the tense part of the tympanic membrane, with no stem cells in the central part of the tense part. In the appropriate culture medium, stem cells proliferated well and could be initially purified by apposition time.