Previous studies have shown that Acetylcholine (ACh) evokes an increase in short circuit current (I_sc) across porcine tracheal epithelia and that the majority of this increase results from stimulation of Cl^–secretion. Forskolin is known to evoke anion secretion by activation of CFTR via elevation of [cAMP]_i levels. The mechanism by which ACh acts is unclear therefore the aim of this study was to investigate this mechanism. Trachea were removed from humanely killed pigs and mounted in Ussing chambers where effects of these secretagogues upon spontaneous current could be explored. Exposure to forskolin evoked an increase in I (4.0±1.7µAcm^-2, n=7) (mean ± sem) and this wasnot significantly different than tissue pretreated with ACh (4.8±1.7, n=7). Application of ACh evoked a larger increase in I_sc (12.5±2.4, n=7) which was unaffected by pre-treatment of forskolin (11.0.±1.2, n=7). Pretreatment with bumetanide (100µM), the basolateral NaK2Cl triple cotransporter inhibitor, significantly decreased baseline I (15.6±1.8%, n=9 (paired t-test, p<0.05)), it also significantly inhibited the response to forskolin (58.8±10.1%, n=9) and ACh following pre-treatment with forskolin (66.5±17.9%, n=7). The response to ACh was also significantly inhibited by bumetanide (92.2±5.1%, n=5 (t-test)). In the presence of amiloride, the Na⁺ channel inhibitor (10µM), the baseline was significantly decreased (39.9±6.4%, n=7) but the responses to forskolin and ACh following pre-treatment with forskolin were unaffected. Together these data suggest that forskolin and ACh employ different mechanisms in the regulation of anion secretion, neither of which involve Na⁺ transport but are both dependent (at least in part) upon uptake of Cl^–across the basolateral membrane via the NaK2Cl cotransporter. It may be that these secretagogues act on different cell types; forskolin may act on the surface epithelia whereas ACh acts on the submucosal glands. It is also possible that ACh activates basolateral K⁺ channels by increasing [Ca²⁺]_i levels, but these hypothesis requires further investigation.