Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M₂ muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (PO₂ = 30 and 40 Torr) from the point of fertilization. In hypoxic fish the heart rate was significantly lower than in normoxic controls from 2 to 10 days post fertilization (dpf). At the more severe level of hypoxia (30 Torr), there were significant increases in the relative mRNA expression of M₂ and the cardiac type -adrenergic receptors (1AR, 2aAR, and 2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at PO₂ = 40 Torr) or significantly attenuated (at PO₂ = 30 Torr) in larvae experiencing M₂ receptor knockdown (using Morpholino antisense oligonucleotides). Sham injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimes. The expression of 1AR, 2aAR, 2bAR, and M₂ mRNA were all altered at various stages between 1 and 4 dpf in larvae experiencing M₂ knockdown. Interestingly, M₂ receptor knockdown unveiled a cardioinhibitory role for the ₂-adrenergic receptor. This is the first study to demonstrate a specific role of the M₂ muscarinic receptor in the initiation of hypoxic bradycardia in fish.