The effects of 5-hydroxytryptamine (5-HT) and calcitonin gene-related peptide (CGRP), which are colocalized in nerve terminals in the airway, on Cl^– secretion in rat tracheal epithelia were tested. Short-circuit current (I _sc) was measured after rat tracheal epithelial monolayers were cultured on porous filters. In rat tracheal monolayers 5-HT and CGRP increased I _sc upon addition to the serosal compartment, in a dose-dependent manner with EC₅₀ values at 5 μmol/l and 5 nmol/l, respectively. The responses were dependent on the presence of Cl^– in the bathing solution and were inhibited by 100 μmol/l bumetanide. When 5-HT or CGRP was added after the administration of forskolin, the responses were not observed. 5-HT and CGRP increased the intracellular cAMP concentration. Low-Ca²⁺ buffer (0.1 mmol/l) and pretreatment with BAPTA/AM (10 μmol/l), thapsigargin (1 μmol/l) or indomethacin (10 μmol/l) did not affect the responses to 5-HT and CGRP. The 5-HT-induced response was not inhibited by 5-HT₂ and/or 5-HT₄ antagonists. These results indicate that in the rat tracheal epithelia 5-HT and CGRP increase Cl^– secretion by an increase in intracellular cAMP concentration via direct activation of basolateral receptors, and that the response to 5-HT is not mediated via 5-HT₄ receptors.