Oxygen chemoreceptors elicit cardiorespiratory reflexes in all vertebrates but consensus on oxygen sensing-signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H₂S) metabolism is involved in O₂ sensing in vascular smooth muscle. Here, we examined the possibility that H₂S is an O₂ sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O₂ sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H₂S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H₂S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H₂S synthesizing enzymes, CBS and CSE, was present in branchial tissue. Homogenized gills produced H₂S enzymatically and H₂S production was inhibited by O₂, whereas mitochondrial H₂S consumption was O₂-dependent. Ambient hypoxia did not affect plasma H₂S in unanesthetized trout, but produced a PO₂-dependent increase in a sulfide moiety suggestive of increased H₂S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H₂S produced a similar ~10 mV depolarization. These studies are consistent with H₂S involvement in O₂ sensing/signal transduction pathway(s) in chemoreceptive cells as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby [H₂S] is governed by the balance between constitutive production and oxidation, tightly couples tissue [H₂S] to PO₂ and may provide an exquisitely sensitive, yet simple, O₂ sensor in a variety of tissues.