A method for middle ear epithelial (CMEE) cell culture with active mucus secretory function has been successfully developed, using the chinchilla as an animal model. CMEE cells were dissociated by protease digestion from the middle ear mucosa. The CMEE cells grown in primary culture incorporated [3H]glucosamine into a glycoconjugate after its release into medium. This substance was characterized biochemically as mucin, although the production of mucin by the cells required growth on a substratum of collagen gel. These cultures provide an excellent model for studying factors that regulate synthesis and secretion of glycoproteins in CMEE cells.