Experiments carried out in conscious guinea pigs suggest that citric acid evoked coughing is partly mediated by TRPV1 receptor-dependent activation of tachykinin-containing, capsaicin-sensitive C-fibers. In vitro electrophysiological analyses indicate, however, that acid also activates capsaicin-sensitive and -insensitive vagal afferent nerves by a TRPV1-independent mechanism, and studies in anesthetized guinea pigs show that coughing evoked by acid is mediated by activation of capsaicin insensitive vagal afferent nerves. In the present study we have characterized the mechanisms of citric acid evoked coughing in anesthetized guinea pigs. Drugs were administered directly to the Krebs buffer perfusing the extrathoracic trachea. Citric acid was applied topically to the tracheal mucosa, directly into the tracheal perfusate in increasing concentrations and at 1 minute intervals. Citric acid dose-dependently evoked coughing in anesthetized guinea pigs. This was mimicked by hydrochloric acid but not by sodium citrate. The coughing evoked by acid was nearly or completely abolished by tetrodotoxin or by cutting the recurrent laryngeal nerves. Perfusing the trachea with a low chloride buffer potentiated the acid-induced cough reflex. In contrast, prior capsaicin desensitization, 10µM capsazepine, Ca++-free perfusate, 0.1µM iberiotoxin, 1µM atropine, 10µM isoproterenol, 10µM albuterol, 3µM indomethacin, 0.1µM HOE 140, a combination of neurokinin1 (NK₁; CP99994), NK₂ (SR48968) and NK₃ (SB223412) receptor antagonists (0.1µM each), a combination of histamine H₁ (3µM pyrilamine) and cysLT₁ (1µM ICI198615) receptor antagonists, superior laryngeal nerve transection or epithelium removal did not inhibit citric acid evoked coughing. These and other data indicate that citric acid evoked coughing in anesthetized guinea pigs is mediated by direct activation of capsaicin insensitive vagal afferent nerves perhaps through sequential activation of acid sensing ion channels and chloride channels.