Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl⁻ secretion and an inverse response of the short-circuit current (I _sc) toward stimulation with carbachol (CCh). Alternative Cl⁻ channels are found in airway epithelia and have been attributed to residual Cl⁻ secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversedI _sc upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca²⁺-dependent Cl⁻ conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage andI _sc were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I _sc in CF rectal biopsies but caused a negative I _sc in non-CF subjects. The CCh-induced negative I _sc in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positiveI _sc was significantly enhanced in CF and was caused by activation of a luminal K⁺ conductance, as shown by the use of the K⁺ channel blockers Ba²⁺ and tetraethylammonium. Moreover, a cAMP-dependent luminal K⁺conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl⁻ channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl⁻conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca²⁺- and cAMP-dependent K⁺ secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.