We performed intracardiac electrophysiologic studies of the effects of vasoactive intestinal peptide (VIP, 0.125 μg/kg/min) on sinus and atrioventricular (AV) nodal function, intracardiac conduction, and myocardial refractoriness in two groups of neonatal dogs (aged 6–16 d). Group I consisted of eight neonates in whom VIP was administered after bilateral vagotomy and β-blockade with propranolol. Group II consisted of five neonates studied after vagotomy and propranolol, plus total chemical sympathectomy (6-hydroxydopamine). In both groups, VIP resulted in a significant shortening of sinus cycle length. AV nodal conduction time, measured as the AH interval (the time from the onset of the atrial electogram to the onset of the His bundle electrogram in the His electrode catheter) during atrial pacing, also shortened after VIP. His-Purkinje conduction time and atrial effective refractory periods were unchanged by VIP. In other experiments, the direct chronotropic effect of VIP was evaluated in five isolated neonatal canine hearts using a modified Langendorff technique. In these hearts, the spontaneous cycle length decreased from 403 ± 88 to 293 ± 69 ms, or −28 ± 4% (mean ± SD), after exposure to 0.1–0.5 nmol of VIP (p < 0.001). In nine other newborns (aged 4–16 days), the effect of selective α1- and α2-adrenergic receptor blockade on the positive chronotropic effect of VIP was evaluated. The effect of VIP on sinus cycle length was not altered by the α2-adrenergic receptor blocker prazosin or by the α2-adrenergic receptor blocker yohimbine. These data indicate that, in the neonatal dog, VIP shortens sinus cycle length and AV nodal conduction time but has no effect on infranodal conduction or myocardial refractoriness. We find no evidence that VIP's chronotropic effect is modified by α-adrenergic receptor blockade. VIP may play a role in the neural modulation of heart rate and AV nodal conduction in the neonate.