This study was carried out to determine the effect of protease-activated receptor-2 (PAR2) activation on the pulmonary chemoreflex responses and on the sensitivity of isolated rat vagal pulmonary chemosensitive neurons. In anaesthetized, spontaneously breathing rats, intratracheal instillation of trypsin (0.8 mg ml(-1), 0.1 ml), an endogenous agonist of PAR2, significantly amplified the capsaicin-induced pulmonary chemoreflex responses. The enhanced responses were completely abolished by perineural capsaicin treatment of both cervical vagi, suggesting the involvement of pulmonary C-fibre afferents. In patch-clamp recording experiments, pretreatment with trypsin (0.1 microM, 2 min) potentiated the capsaicin-induced whole-cell inward current in isolated pulmonary sensory neurons. The potentiating effect of trypsin was mimicked by PAR2-activating peptide (PAR2-AP) in a concentration-dependent manner. PAR2-AP pretreatment (100 microM, 2 min) also markedly enhanced the acid-evoked inward currents in these sensory neurons. Furthermore, the sensitizing effect of PAR2 was completely abolished by pretreatment with either U73122 (1 microM, 4 min), a phospholipase C inhibitor, or chelerythrine (10 microM, 4 min), a protein kinase C (PKC) inhibitor. In summary, our results have demonstrated that activation of PAR2 upregulates the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro, and this effect of PAR2 activation was mediated through the PKC-dependent transduction pathway. These results further suggest that the hypersensitivity of these neurons may play a part in the development of airway hyper-responsiveness resulting from PAR2 activation.