This study was carried out to investigate the role of cationic charge in the hypersensitivity of pulmonary C-fibers induced by airway exposure to synthetic cationic protein poly-L-lysine (PLL) in anesthetized rats. Inhalation of PLL aerosol induced a distinctly irregular breathing pattern, and significantly enhanced the pulmonary chemoreflex responses to capsaicin. However, after the cationic charges were completely removed from PLL by succinylation, the succinylated PLL no longer produced any change in either the baseline breathing pattern or the reflex responses to capsaicin. In addition, the effects of PLL were also abolished after premixing it with a polyanion, poly-L-glutamic or poly-L-aspartic acid, before delivery. In sharp contrast, when delivered within 5 min after the PLL aerosol, these two polyanions were completely ineffective in reversing the effects of PLL. Electrophysiological recording of the afferent activity of single pulmonary C-fibers further supported our conclusion that the cationic charge carried by this protein is primarily responsible for generating the stimulatory and sensitizing effects of PLL on these afferents.