Our studies of fasted anesthetized rats have shown that all spontaneous relaxations of the antrum are nitric oxide (NO) dependent. Duodenal motility is patterned into propagating "grouped" motor activity interposed with "intergroup" periods of nonpropagating motor activity; in the duodenum, only intergroup relaxations are NO dependent. We examined the involvement of NO and ATP in spontaneous motor activities of the gastroduodenum in vivo: contractions and relaxations were recorded and analyzed simultaneously from the antrum (S₁) and proximal duodenum (D₁) of anesthetized Sprague-Dawley rats (n = 10/group), using extraluminal foil strain gauges. Treatment with the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral and intergroup relaxations, whereas grouped relaxations were enhanced (P < 0.05). These effects were reversed with L-arginine (300 mg/kg iv). L-NAME also increased (P < 0.05) the amplitude of duodenal contractions. ATP (8 mg · kg¹ · min¹ iv) stimulated relaxations at S₁ and D₁ that were blocked by the P₂-purinoceptor antagonist suramin (60 mg/kg iv). This treatment did not affect spontaneous antral relaxations; however, duodenal grouped relaxations were attenuated. Desensitization to the P_2x-purinoceptor agonist ,-methylene ATP (300 µg/kg iv) gave results similar to suramin. In contrast, the P_2y-purinoceptor agonist 2-methylthio-ATP (2-MeS-ATP; 360 µg/kg iv) evoked duodenal relaxations that were attenuated by L-NAME, and desensitization to 2-MeS-ATP attenuated intergroup relaxations. Spontaneous relaxations of the rat antrum and duodenal intergroup relaxations are NO dependent. Both gut regions relax in response to systemically administered ATP; this response is sensitive to suramin. Grouped duodenal relaxations display functional sensitivity to suramin and P_2x- purinoceptor desensitization, indicative of the involvement of ATP and P_2x purinoceptors. P_2y purinoceptors must also be present; however, these occur on elements releasing NO. Although NO does not mediate grouped relaxations or duodenal contractions, the sensitivity of these responses to L-NAME indicates that the pathway(s) controlling these responses is modulated by NO.