1 An experimental set up is used whereby the serosal (out)side or mucosal (in)side of the guinea‐pig isolated tracheal tube can be stimulated selectively with drugs and reactivity measured. 2 Potassium induces a concentration‐dependent (5 − 70 mM) monophasic contraction of tracheal tubes when added on the outside. In contrast, on the inside, potassium induces a concentration‐dependent relaxation at low concentrations (5–40 mM) which was reversed into a contraction up to approximately basal tone at higher concentrations (50–70 mM). 3 Epithelium denudation reversed the potassium‐induced relaxation into a contraction. Interestingly, in the ‘half epithelium‐denuded trachea the contractions were significantly (P<0.01) reduced by 46% compared to complete epithelium‐denuded tissues. 4 Incubation with the nitric oxide (NO) synthase inhibitor NΩ‐nitro‐L‐arginine methyl ester (l‐NAME, 120 μm) for 30 min on the inside of the tracheal tube completely prevented the relaxation. However, L‐NAME did not reverse the potassium‐induced relaxation into a contraction. This indicates that potassium does not penetrate through the epithelial layer. 5 It is concluded that depolarization of smooth muscle cells leads to a monophasic contraction and that depolarization of the epithelium leads to a relaxation of tracheal smooth muscle. The epithelial layer has an important barrier function and can release relaxing factors like NO.