Serotonin (5-HT) type 3 receptor (5-HT₃-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT₃-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT₃-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT₃-R) and confocal microscopy localized 5-HT₃-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT₃-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5-150 µM) and 5-HT₃-R agonist 2-methyl-5-HT (5-150 µM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 ± 5.9%) by the specific 5-HT₃-R antagonist ICS-205-930 (50 µM), whereas katanserin and p-4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca²⁺ and Mg²⁺ in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT₃-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.