This study investigated: 1) whether pulmonary C-fibers are activated by a transient increase in the CO₂ concentration of alveolar gas; 2) if the CO₂ sensitivity of these afferents is altered during airway inflammation. Single-unit pulmonary C-fiber activity was recorded in anesthetized, open-chest rats. Transient alveolar hypercapnia (HPC) was induced by administering a CO₂-enriched gas mixture (25-30% CO₂, 21% O₂, balance N₂) for 5-8 breaths, which increased alveolar CO₂ concentration progressively to near or above 13% for 3-5 s and lowered the arterial pH transiently to 7.10 ± 0.05. Our results showed: 1) HPC evoked only a mild stimulation in a small fraction (4/47) of pulmonary C-fibers, and there was no significant change in fiber activity (FA = 0.22 ± 0.16 impulses/s; P > 0.1, n = 47); 2) in sharp contrast, after airway exposure to poly-L-lysine (PLL), a cationic protein known to induce mucosal injury, the same challenge of transient HPC activated 87.5% of the pulmonary C-fibers tested and evoked a distinct stimulatory effect on these afferents (FA = 6.59 ± 1.78 impulses/s; P < 0.01, n = 8); 3) similar potentiation of the C-fiber response to HPC was also observed after acute exposure to ozone (n = 6), and during a constant infusion of inflammatory mediators such as adenosine (n = 15) or prostaglandin E₂ (n = 12); and 4) The enhanced C-fiber sensitivity to CO₂ after PLL was completely abrogated by infusion of NaHCO₃ (1.82 mmol/kg/min) that prevented the reduction in pH during HPC (n = 6). In conclusion, only a small percentage (< 10%) of the bronchopulmonary C-fibers exhibit CO₂ sensitivity under control condition, but alveolar HPC exerts a consistent and pronounced stimulatory effect on the C-fiber endings during airway inflammation. This effect of CO₂ is probably mediated through the action of hydrogen ions.