To determine whether intrinsic cardiac neurons are sensitive to oxygen-derived free radicals in situ, studies were performed in 44 open-chest anesthetized dogs. 1) When H2O2 (600 μM) was administered to right atrial neurons of 36 dogs via their local arterial blood supply, neuronal activity either increased (+92% in 16 dogs) or decreased (−61% in 20 dogs), depending on the population of neurons studied. H2O2 (600 μM) administered into the systemic circulation did not affect neuronal activity, measured cardiac indexes, or aortic pressure. 2) The iron-chelating agent deferoxamine (20 mg/kg iv), a chemical that prevents the formation of oxygen-derived free radicals, reduced the activity generated by neurons (−57%) in 8 of 10 dogs. 3) H2O2 did not affect neuronal activity when administered in the presence of deferoxamine in these 10 dogs. 4) When the ATP-sensitive potassium (KATP) channel opener cromakalim (20 μM) was administered to intrinsic cardiac neurons in another 21 animals via their regional arterial blood supply, ongoing neuronal activity in 15 of these dogs decreased by 54%. 5) Neuronal activity was not affected by H2O2 when administered in the presence of cromakalim in 16 dogs. These data indicate that 1) some intrinsic cardiac neurons are sensitive to exogenous H2O2, 2) such neurons are tonically influenced by locally produced oxygen-derived free radicals in situ, and 3) intrinsic cardiac neurons possess KATP channels that are functionally important during oxidative challenge.