We tested the hypothesis that endogenously produced hydrogen sulfide (H₂S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water PO₂= 35 mm Hg), plasma H₂S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney), produced H₂S in vitro when incubated with L-cysteine. In both tissues, the production of H₂S was eliminated by adding the cystathionine -synthase (CBS) inhibitor, aminooxyacetate (AOA). CBS and cystathionine -lyase (CSE) were cloned and sequenced and the results of real time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H₂S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 X 10^-6 M) or depolarizing levels of KCl (10 mM) caused secretion of catecholamines without altering H2S output suggesting that neuronal excitation is required for H₂S release. Addition of H₂S (at concentrations exceeding 5 X 10^-7 M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca²⁺-free perfusate was used. These data, together with the finding that H₂S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H₂S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca²⁺ mediated exocytosis.