To determine whether the excitabilities of pulmonary C fibers to chemical and mechanical stimuli are altered by CO₂-induced acidosis, single-unit pulmonary C-fiber activity was recorded in anesthetized, open-chest rats. Transient alveolar hypercapnia (HPC) was induced by administering CO₂-enriched gas mixture (15% CO₂, balance air) via the respirator inlet for 30 s, which rapidly lowered the arterial blood pH from a baseline of 7.40 ± 0.01 to 7.17 ± 0.02. Alveolar HPC markedly increased the responses of these C-fiber afferents to several chemical stimulants. For example, the C-fiber response to right atrial injection of the same dose of capsaicin (0.25-1.0 µg/kg) was significantly increased from 3.07 ± 0.70 impulses/s at control to 8.48 ± 1.52 impulses/s during HPC (n = 27; P < 0.05), and this enhanced response returned to control within ~10 min after termination of HPC. Similarly, alveolar HPC also induced significant increases in the C-fiber responses to right atrial injections of phenylbiguanide (4-8 µg/kg) and adenosine (0.2 mg/kg). In contrast, HPC did not change the response of pulmonary C fibers to lung inflation. Furthermore, the peak response of these C fibers to capsaicin during HPC was greatly attenuated when the HPC-induced acidosis was buffered by infusion of bicarbonate (1.36-1.82 mmol · kg¹ · min¹ for 35 s). In conclusion, alveolar HPC augments the responses of these afferents to various chemical stimulants, and this potentiating effect of CO₂ is mediated through the action of hydrogen ions on the C-fiber sensory terminals.