Key pointsr Hydrogen sulphide (H 2 S), a gaseous neurotransmitter, is involved in oxygen sensing in glomus cells, which are oxygen-sensing cells found in the mammalian carotid body.r Neuroepithelial cells (NECs) are oxygen-sensing cells of fish and are thought to be phylogenetic precursors of mammalian glomus cells; however, the oxygen-sensing mechanisms of these cells remain largely unknown.r Both adult and larval zebrafish responded to exogenous H 2 S by increasing ventilation in a dose-dependent manner; H 2 S increased intracellular [Ca 2+ ] in NECs.r Inhibiting endogenous H 2 S production decreased or abolished the ventilatory response to hypoxia in both adult and larval zebrafish.r The results demonstrate an important role for H 2 S in oxygen sensing in zebrafish.Abstract The current study investigated the role of hydrogen sulphide (H 2 S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na 2 S), an H 2 S donor. In vertebrates, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H 2 S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na 2 S to the water partially rescued the effects of inhibiting endogenous H 2 S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 μM Na 2 S, supporting a role for H 2 S in Ca 2+ -evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H 2 S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response.