Airway hyperresponsiveness is a cardinal feature of asthma. Lung C-fiber activation induces central and local defense reflexes that may contribute to airway hyperresponsiveness. Initial studies show that substance P (SP) activates C-fibers even though it is produced and released by these same C-fibers. SP may induce release of other endogenous mediators. Bradykinin (BK) is an endogenous mediator that activates C-fibers. The hypothesis was tested that SP activates C-fibers via Bk release. Guinea pigs were anesthetized and C-fiber activity (FA), pulmonary insufflation pressure (PIP), heart rate and arterial blood pressure were monitored before and after intravenous injection of capsaicin (Cap), SP and Bk. Identical agonist challenges were repeated after infusion of an antagonist cocktail of des Arg⁹, [Leu⁸] Bk (10^-3 M, B1 antagonist) and Hoe 140 (10^-4 M, B2 antagonist). After antagonist administration Bk increased neither PIP nor FA. Increases in neither PIP nor FA were attenuated after Cap or SP challenge. In a second series of experiments Cap and SP were injected before and after infusion of indomethacin (1 mg/Kg, i.v.) to determine if either agent activates C-fibers through release of arachidonic acid metabolites. Indomethacin administration decreased the effect of SP challenge on FA but not PIP. The effect of Cap on FA or PIP was not altered by indomethacin. In subsequent experiments C-fibers were activated by prostaglandin E₂ and F₂. Therefore exogenously applied SP stimulates an indomethacin sensitive pathway leading to C-fiber activation.