The current model of Cl^– secretion in mammalian colonic crypts involves electroneutral Cl^– entry across the basolateral membrane, facilitated by Na⁺-K⁺-2Cl^– co-transport, followed by electrogenic Cl^– exit across the apical membrane through cAMP activated Cl^– channels. Simultaneous activation of basolateral K⁺ channels is thought to maintain a favourable electrical gradient for apical Cl^– secretion by limiting cell depolarisation. In this study we have sought evidence for K⁺ channel activation by cAMP using perforated whole cell patch clamp in human colonic crypts. Sigmoid biopsies were obtained from patients undergoing routine colonoscopy for altered bowel habit. Colonic crypts were isolated using a Ca²⁺ chelation technique as described previously (Bowley et al., 2003). Slow-whole cell currents (with amphotericin B in the pipette) were measured from colonocytes in the mid-third of intact crypts. Single channel currents were measured from the basolateral membrane using the cell attached patch technique. Data are presented as mean ± 1 SEM, with n, the number of experiments. Comparison was by paired t test with significance assumed at p < 0.05. Under basal conditions, whole cell currents were predominantly K⁺ selective with a conductance (G) of 0.98 ± 0.1 nS and a reversal potential (E_rev) of -62 ± 2 mV (n = 40). Forskolin (FSK, 10 μM) gave a ~3 fold increase in G and a depolarising shift in E_rev of 29.8 ± 7 mV (n = 8, P < 0.05). The FSK-stimulated conductance was significantly inhibited by removal of bath Cl^– (107 ± 3%, n = 8), or addition of NPPB (200 μM; 85 ± 18%, n = 4) but not DIDS (100 μM; 1 ± 8%, n = 4), indicating activation of CFTR. The effect of FSK on GK⁺ was investigated under low Cl^– conditions (4mM Cl^– in bath and pipette), where FSK reduced the basal conductance from 1.43 ± 0.6 nS to 0.56 ± 0.1 nS (n = 6, P < 0.05) and depolarised the cell from -62 ± 4 to -44 ± 8 mV (P < 0.05), consistent with K⁺ channel inhibition. Addition of chromanol 293B (10 μM), an inhibitor of the cAMP-activated K⁺ channel KCNQ1, to FSK-stimulated crypts, was without effect in both high and low Cl^– experiments. Single channel analysis of the basolateral membrane revealed that intermediate conductance Ca²⁺-sensitive K⁺ channels (IK_Ca) were inhibited by forskolin (n = 7, p<0.05). In conclusion, FSK stimulated a Cl^– conductance with pharmacological properties consistent with CFTR. We found no evidence of K⁺ channel activation by cAMP in human colonic crypts. Paradoxically, FSK inhibited the basal whole cell K⁺ conductance and basolateral IK_Ca channels suggesting that basolateral K⁺ channels have a permissive, yet limiting, role in Cl^– secretion in human colonic crypts.