We used the short-circuit current (Isc) and patch-clamp techniques to investigate the effects of methoxsalen (MTX) on the electrogenic Cl− secretion of the mouse jejunum. MTX stimulated a sustained increase in Isc that was dose dependent. Bumetanide inhibited MTX-stimulated Isc in a dose-dependent manner consistent with activation of Cl− secretion. MTX failed to stimulate Isc following maximal activation of the cAMP pathway by forskolin, but did increase Isc after a submaximal dose of forskolin. Glibenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR), reduced the MTX-stimulated increase of Isc by 59 ± 6%. The cAMP-dependent K+ channel blocker 293B did not alter the MTX-activated Isc; however, clotrimazole, an intermediate Ca2+-activated K+ channel (IKCa) blocker, reduced the MTX-stimulated Isc. MTX did not alter Na+–glucose cotransport across the mouse jejunum.In cell-attached membrane patches, MTX increased the open probability of the basolateral IKCa channel of isolated crypts. These data suggest that the CFTR and IKCa channels participate in the MTX-activated, sustained Cl− secretory response of the mouse jejunum.