The purpose of this study was to determine whether epidermal growth factor (EGF) regulates mucous differentiation of airway epithelial cells in culture. Reduction of the EGF concentration below 25 ng/ml, which is the concentration routinely used in rat tracheal epithelial cell cultures, resulted in a 2 to 3-fold decrease in the percentage of mucous cells as determined by a mucin monoclonal antibody. The amount of secreted mucin decreased more than 10-fold within 5 days after reducing the EGF concentration. MUC5 gene expression, which was previously shown to correlate with mucous differentiation, was also reduced more than 8-fold. Addition of 25 ng/ml EGF to EGF-deprived cultures resulted in rapid induction of MUC5 expression. Following tissue injury and during inflammation the release of EGF and its functional analogue TGF alpha have been observed and may be involved in the mucus hypersecretion characteristic of many airway diseases.