Diarrheagenic strains of Escherichia coli have been shown to produce a heat-stable enterotoxin (ST) that simulates guanylate cyclase, increases short-circuit current (Isc), and inhibits active Cl absorption in the intestine. In rabbit ileum, the ion transport effects are smaller than those produced by cAMP-related agonists. Because ST may be a selective cGMP agonist, we further explored its mode of action in rabbit ileum. ST inhibits net Na and net Cl absorption. ST also inhibits the same fraction of Cl influx across the brush border that theophylline inhibits. At maximal doses, ST and 8-bromo-cGMP (8-Br-cGMP) had nearly equal, nonadditive effects of Isc that were about 66% of that produced by 8-Br-cAMP. ST increased mucosal cGMP concentration 16-fold, whereas epinephrine, an inhibitor of secretion, increased cGMP concentration by only 30%. This is insufficient to alter ion transport because doses of ST that increased cGMP concentration by 100% failed to alter Cl fluxes. Furthermore, epinephrine did not increase cGMP concentration in isolated enterocytes. We conclude that 1) cGMP mediates ST effects on ion transport, and 2) although ST and cAMP-related agonists have the same antiabsorptive effects, ST is less effective in stimulating electrogenic Cl secretion.