Cl⁻secretion in the colon can be activated by an increase of either intracellular Ca²⁺ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca²⁺concentration ([Ca²⁺]_i), activated a transient lumen-negative equivalent short-circuit current (I _sc) [change (Δ) inI _sc = −79.4 ± 7.5 μA/cm²]. Previous studies indicated that intracellular Ca²⁺ directly acts on basolateral K⁺ channels, thus enhancing driving force for luminal Cl⁻ exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (ΔI _sc = −42.4 ± 7.2 μA/cm²) that was inhibited by basolateraltrans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (ΔI _sc = 167.7 ± 32.7 μA/cm²), suggesting cooperativity of the Ca²⁺- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positiveI _sc was probably due to activation of apical K⁺channels because it was blocked by luminal Ba²⁺ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I _sc. Under these conditions, CCH induced a large further increase in lumen-negativeI _sc(ΔI _sc = −100.0 ± 21.0 μA/cm²). We conclude that CCH acting via [Ca²⁺]_ican induce Cl⁻ secretion only in the presence of cAMP, i.e., when luminal Cl⁻ channels are already activated. The activation of a luminal and basolateral K⁺ conductance by CCH may be essential for transepithelial KCl secretion in human colon.