Abstract The effect of serotonin on capsaicin-sensitive vagal C-fibre afferent nerves was evaluated in an ex vivo vagally innervated mouse lung preparation. Action potentials arising from receptive fields in the lungs were recorded with an extracellular electrode positioned in the nodose/jugular ganglion. Among the 62 capsaicin-sensitive C-fibres studied (conduction velocity ∼0.5 m s(-1)), 71% were of the nodose phenotype and 29% of the jugular phenotype. The nodose C-fibres responded strongly to serotonin and this effect was blocked with the 5-HT3-receptor antagonist ondansetron. Using single cell RT-PCR, we noted that the vast majority of nodose neurons retrogradely labelled from the lung, expressed 5-HT3 receptor mRNA. The jugular C-fibres also responded strongly to serotonin with action potential discharge, but this effect was not inhibited by ondansetron. Lung-specific jugular neurons did not express 5-HT3 receptor mRNA but frequently expressed 5-HT1 or 5-HT4 receptor mRNA. Mast cells are the major source of serotonin in healthy murine airways. Ovalbumin-induced mast cell activation in actively sensitized lungs caused action potential discharge in jugular but not nodose C-fibres. The data show that vagal C-fibres in the respiratory tract of the mouse are strongly activated by serotonin. Depending on the C-fibre subtype both 5-HT3 and non-5-HT3 mechanisms are involved.