This study investigated enteric nervous system (ENS) involvement in intestinal secretion induced by cyclic GMP-dependent secretagogues. The investigation was based upon the study of transepithelial ion transport in rat small and large intestine and in guinea pig caecum using voltage-clamped in vitro preparations mounted in Ussing chambers. ENS participation was established from the use of neural blocking agents (tetrodotoxin (TTX), bicuculline and capsaicin). The relative contribution of the myenteric plexus was assessed by selectively stripping tissues of the longitudinal muscle layer. All tissues, both unstripped and stripped, responded to <I>Escherichia coli </I>STa enterotoxin, guanylin and the nitric oxide (NO) donor sodium nitroprusside (SNP) with a dose-dependent increase in inward short circuit current (I_SC). Regarding STa/guanylin, TTX inhibited this I_SCin unstripped rat distal colon, ileum and guinea pig caecum, demonstrating that the ENS plays an important role in these tissues. In rat distal colon, TTX induced an abolition of the STa/guanylin response in both preparations, indicating submucous plexus involvement. In rat proximal colon there was no discernible TTX-sensitive component observed. The ileum displayed partial control from both the myenteric and submucous plexuses, whereas the caecum exhibited partial control from the myenteric plexus alone. Bicuculline inhibited STa action to a significant degree in the caecum while capsaicin inhibited secretion in the proximal colon. In rat small intestine, the SNP-induced I_SC was inhibited by TTX in both unstripped and stripped tissues. In contrast, inhibitory pathways were shown to exist in distal colon exposed to SNP, TTX revealing an enhancement of SNP-induced secretion in the stripped preparation. Thus, although there is clear involvement of the ENS in the actions of STa/guanylin and SNP, it is not possible to make a general statement regarding its contribution throughout the length of the alimentary canal due to the extent of inter segmental and inter species variations.