To evaluate the role of nitric oxide (NO) in the flow response after brief coronary arterial occlusion, NO formation by the isolated guinea pig heart was assessed by a specific difference spectrophotometric assay. Release of NO under basal conditions was 121.8±10.5 pmol×min−1and increased to 211.1±16.8 pmol×min−1after 60 seconds of coronary occlusion. Simultaneously, release of cGMP and adenosine increased by 87% and 652%, respectively. The kinetics of NO release paralleled the reactive hyperemic flow response. Inhibition of NO synthesis with nitro-l-arginine methyl ester (l-NAME, 30 μM) significantly reduced basal flow and attenuated reactive hyperemia, flow repayment, and repayment ratio. l-NAME decreased release of cGMP but significantly increased adenosine release under basal conditions and during reactive hyperemia. Oxyhemoglobin (5 μM) potentiated the effects of l-NAME. The stereoisomer nitro-d-arginine methyl ester was ineffective. Our results suggest 1) NO is an important regulator of coronary flow during reactive hyperemia as well as under basal flow conditions and 2) the significance of the increased adenosine release when NO synthesis is inhibited remains to be determined.