Carbon monoxide (CO) is a gaseous signalling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio rerio. Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive neuroepithelial cells (NECs) of larvae (4 days post fertilization) and adults indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water PO₂ of 30 mm Hg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX (ZnPPIX) treatment in adults) exhibited increased ventilation frequency (V_f) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting V_f in fish with diminished CO production and in some cases (e.g. hypoxic sham larvae) CO modestly reduced V_f below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-haemoglobin interactions which might influence ventilation. PHZ treatment did not cause changes in V_f of normoxic larvae and the addition of CO to PHZ-exposed larvae resulted in a significant decrease in sham and HO-1 deficient fish under normoxic conditions. This study demonstrates for the first time that CO plays an inhibitory role in the control of breathing in larval and adult zebrafish.