ACh induces anion secretion in intact porcine tracheal epithelia (Constable et al 2003) and isolated submucosal glands (Yang et al 1988) but the mechanism of activation is unclear. ACh and the influx of Ca²⁺ resulted in Cl^– secretion across the apical membrane of isolated porcine tracheal submucosal glands (Yang et al 1988). ACh may therefore act to increase [Ca²⁺]_i, activating basolateral Ca²⁺-activated K⁺ (K_Ca) channels, thus providing an electrical driving force for Cl^– exit across the apical membrane. Basolateral K⁺ channels have been demonstrated in several respiratory epithelia and have been shown to be involved in anion secretion (McCann and Welsh 1990). Here we investigate the role of basolateral K⁺ channels in the ACh-induced anion secretion in porcine tracheal epithelium. Tracheas were removed from humanely killed pigs and the epithelial layer dissected from underlying cartilage. Epithelial sheets were mounted on Ussing chambers and bathed in HCO₃^– buffered solution, gassed with 95%O2/5%CO2 at 37°C. ACh evoked a peak increase in I_sc (17.6±1.3μAcm^-2, mean ± S.E.M., n=6) that slowly decayed over time. In the presence of the non-specific K⁺ channel inhibitor barium the response to ACh was significantly inhibited (40.4±9.1%, n=6, student’s paired t-test used throughout, p=0.05 taken as threshold of significance), indicating a role for K⁺ channels in this process. Specific mediators of different K⁺ channels were used to identify which types are involved in the ACh-induced anion secretion. Chromanol 293B, an inhibitor of voltage-gated K⁺ channels had no effect on the response to ACh, suggesting if present, these channels do not play a role in this process. In contrast the K_Ca channel inhibitor clotrimazole significantly inhibited the response to ACh (42.1±7.9%, n=6), indicating a role for K_Ca channels in the ACh-induced anion secretion. In support of this 1-EBIO, a K_Ca channel activator, augments the anion secretion evoked by ACh (63.6±27.1%, n=6). Together these data suggest ACh evokes anion secretion by activating basolateral K_Ca channels, presumably by increasing [Ca²⁺]_i thus driving Cl^– secretion across the apical membrane via anion channels.