In the present study, we investigated whether polycationic peptides affect mucin release from cultured airway goblet cells. Confluent primary hamster tracheal surface epithelial cells were metabolically radiolabeled with [³H]glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of either poly-L-arginine (PLA) or poly-L-lysine (PLL) to assess the effects on [³H]mucin release. Possible cytotoxicity by the polycations was assessed by measuring lactate dehydrogenase release, ⁵¹Cr release, and cell exfoliation. The results were as follows: 1) both PLA and PLL inhibited mucin release in a dose-dependent fashion; 2) there was no significant difference in either lactate dehydrogenase release, ⁵¹Cr release, or the number of floating cells between control and treatment groups; 3) the effects of both PLA and PLL on mucin release were completely blocked by neutralizing the positive charges either by pretreatment with heparin or by N-acetylation of the polycations; and 4) both PLA and PLL completely masked the stimulatory effect of ATP on mucin release. We conclude that these polycationic peptides can inhibit mucin release from airway goblet cells without any apparent cytotoxicity, and the inhibitory effect seems to be attributable to their positive charges. These are the first nonsteroidal agents, to the best of our knowledge, that have been shown to inhibit mucin release from airway goblet cells.