Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H+) is presently lacking. We tested the effects of CO2/H+ alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca2+ imaging method was used to assess CO2/H+-evoked changes in intracellular Ca2+. Exposure to 10 and 20% CO2 or pH 6.8–7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca2+ in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca2+. Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H+-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H+ sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.