Previous neuroanatomical and physiological studies have indicated that nucleus ambiguus (Amb) is one source of vagal motoneurons in the brain stem that innervates the pancreas and which, when stimulated, increases insulin release. To investigate one of the neurotransmitter inputs to Amb neurons and its relation to insulin secretion, bicuculline, a specific γ-aminobutyric acid (GABA) antagonist, was infused bilaterally into the Amb region as well as into a neighboring area, the rostral level of the lateral nucleus tractus solitarius (nts) of anesthetized male rats. Experiments were carried out in the presence or absence of the α-adrenergic blocker phentolamine. In the absence of phentolamine, no increases in plasma insulin levels were seen after bicuculline or vehicle infusion into the Amb region or after bicuculline infusion into the nts, while plasma glucose levels were significantly increased. In the presence of phentolamine, bicuculline infusion into the Amb region led to a prompt and significant increase in plasma insulin levels that could not be accounted for by changes in glycemia. The infusion of vehicle into Amb or of bicuculline into nts produced small or insignificant increases in plasma insulin levels. These results suggest that Amb neurons capable of modulating plasma insulin levels are under tonic GABA inhibition, an effect that appears to be specific for Amb neurons, since bicuculline infusion into another brain stem nucleus (nts) had no effect on insulin release. The fact that phentolamine pretreatment was necessary to reveal the bicuculline-induced effects corroborates previous studies showing that in addition to a central nervous system inhibition of vagal motoneurons by GABA, there is a tonic sympathetic inhibitory input to the endocrine pancreas capable of masking any disinhibition of vagal motoneurons. The physiological role of GABA inhibition of Amb neurons that innervate the pancreatic -cells remains to be determined.