Abstract Neurohumoral agents modulate intestinal transport by interactions with cell membrane receptors. Intracellular second messenger systems implicated in mediation of membrane receptor regulation of cellular events include the phosphoinositide and adenylate cyclase systems. In this study we have investigated the effects of direct postreceptor activation of key components of these systems on intestinal water and electrolyte transport. Rabbit ileal segments (n = 35) were arterially perfused ex vivo with an oxygenated sanguineous solution. The lumen was perfused with an isotonic solution containing 14C-polyethylene glycol as a nonabsorbable marker. Net fluxes of H 2O, Na +, and Cl − in six experimental groups were calculated for three 20-minute periods: basal, drug infusion, and recovery. The control group had no drug infusion. Two phorbol esters—phorbol 12, 13-diacetate (PDA; 10 −5 mol), and phorbol 12, 13-dibutyrate (PDB; 10 −5 mol)—were used to activate protein kinase C, an important component of the phosphoinositide system. The inactive 4α-phorbol 12, 13-didecanoate (PDD; 10 −5 mol) served as a drug-infused control. Forskolin at two doses (FOR; 10 −5 mol and 10 −6 mol) was used to activate adenylate cyclase. The control and PDD groups had no changes in the flux of water and electrolytes. Both PDA and PDB had proabsorptive effects, with the more lipophilic and potent phorbol ester (PDB) having a more pronounced, significant effect (p < 0.05). FOR caused significant secretion of H 2O, Na +, and Cl − in a dose-dependent fashion (p < 0.05). These results indicate that direct protein kinase C activation causes a proabsorptive effect and that direct activation of adenylate cyclase causes a secretory effect in the isolated small bowel. The activation status of these second messenger systems has a major influence on the transport state of the intestine.