A reproducible method is presented for primary cultures of middle ear epithelial cells (MEEC) from chinchillas. The MEEC were first dissociated with protease and grown on collagen-coated membrane using a culture medium containing equal volumes of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 0.5% fetal bovine serum. Outgrowth of cells was first noted within 24 h, reaching confluency in 6-7 days. These cells grew in a monolayer and appeared to be ovoid or polygonal. By immunofluorescence microscopy, these cells stained for cytokeratin, but not for type III collagen. In contrast, fibroblasts stained for type III collagen, but not for cytokeratin. Based on growth characteristics, morphology, and immunofluorescent findings, these cells were determined to be epithelial cells. To retard the outgrowth of fibroblasts, 5 mM putrescine was added to the culture medium on the 2nd day of explant. Contamination with fibroblasts was consistently less than 5% when defined as type III collagen-positive cells. Establishment of a method for the primary culture of MEEC will provide a new approach for studying the role of epithelial cells in the pathogenesis of various types of otitis media.