Spontaneous relaxations occurring within motor activity in the rat gastroduodenum in vivo can be distinguished by their dependence on either nitric oxide (NO) or ATP. We examined the interaction of -aminobutyric acid (GABA) and vasoactive intestinal peptide (VIP) within pathways controlling this activity in the antrum (S) and duodenum (D) of anesthetized Sprague-Dawley rats, using miniaturized extraluminal foil strain gauges oriented perpendicular to (S₁, D₁) or in the axis of (S₂) the circular smooth muscle. The NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral relaxations and, in the duodenum, nonpropagating "intergroup" relaxations. The GABA_A receptor antagonist bicuculline (350 µg/kg sc) had similar effects. The GABA_A agonist 3-amino-1-propanesulfonic acid stimulated L-NAME-sensitive relaxations at S₁ and D₁. Propagating "grouped" responses were unchanged. VIP (6 µg/kg iv) always induced a relaxation of the duodenum, which was attenuated by bicuculline and L-NAME. VIP caused simultaneous responses at S₁ and S₂; however, the antrum displayed either contraction or relaxation in response to VIP. All antral relaxations in response to VIP were attenuated (P < 0.05) by L-NAME; however, only VIP-induced relaxations at S₁ were sensitive to bicuculline. VIP-induced contractions were also unaffected. GABA_A receptors mediate the pathway(s) controlling NO-related spontaneous relaxations of the antrum and duodenal circular muscle. All VIP-induced relaxations are mediated by NO. Spontaneous relaxations of the rat gastroduodenum include responses that involve a GABA_Aergic NO-related pathway, which is targeted by VIP. In addition, VIP can target NO relaxations of the antrum via other pathways.