Interaction between substance P (SP) and PGE₂ on Cl secretion in the guinea pig distal colonic epithelia was investigated. A short-circuit current (I_sc) was measured as an index of ion transport. Mucosa preparations deprived of muscle and submucosa of distal colon were mounted in the Ussing flux chamber and treated with TTX and piroxicam to remove the influences of neuronal activity and endogenous PG synthesis, respectively. Although SP (10⁷ M) itself evoked little increase in I_sc, exogenous PGE₂ concentration dependently enhanced the response of SP. The effect of PGE₂ on the SP-evoked response was mimicked by forskolin and 8-bromoadenosine cAMP. Depletion of Ca²⁺ from the bathing solution reduced the PGE₂-dependent response of SP. Effects of PGE₂, SP, and SP in the presence of PGE₂ on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in isolated crypt cells were measured by the confocal microscope fluorescence imaging system. SP, but not PGE₂, temporally evoked an increase in [Ca²⁺]_i but declined to the baseline within 3 min. A return of the SP-evoked increase in [Ca²⁺]_i was slower in the presence of PGE₂ than SP alone. These results suggest that PGE₂ synergistically enhances SP-evoked Cl secretion via an interaction between the intracellular cAMP and [Ca²⁺]_i in the epithelial cells. In conclusion, SP and PGE₂ could cooperatively induce massive Cl secretion in guinea pig distal colon at epithelial levels.