Despite the importance of GABAergic input to cardiac vagal neurons the electrophysiological properties and possible origins of this innervation have not yet been studied. Individual cardiac vagal neurons were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Cardiac vagal neurons received spontaneous GABAergic inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA(A) receptor antagonist bicuculline. The spontaneous presynaptic GABAergic input to cardiac vagal neurons in the nucleus ambiguus occurred at a significantly lower frequency than that recorded in cardiac vagal neurons in the dorsal motor nucleus of the vagus. To identify a possible source of the GABAergic innervation to cardiac vagal neurons the nucleus tractus solitarius was electrically stimulated. GABAergic synaptic currents in cardiac vagal neurons, in both the dorsal motor nucleus of the vagus (DMNX) and the nucleus ambiguus (NA), were consistently evoked upon stimulation of the nucleus tractus solitarius and these responses were also blocked by bicuculline.