Acetylcholine (ACh) evokes an increase in potential difference (PD) across porcine distal bronchi and both Cl^–and HCOsecretion must be inhibited to block this response (Inglis et al. 1996). This suggests that ACh stimulates secretion of these anions. Serous cells of bronchial submucosal glands are thought to be the main source of anion and fluid secretion with the gland mucous cells being responsible for mucus secretion. Autoradiographic studies have shown expression of M1 and M3 muscarinic receptor subtypes in submucosal glands (Mak & Barnes 1990), but the role of each in mediating ACh-induced secretion of liquid and/or mucus is unclear. The aim of this study is to investigate the effect of blocking either M1 or M3 muscarinic receptors (with Pirenzipine (PZ) and 4-DAMP respectively) on the PD response to ACh, thus indicating the involvement of each subtype in anion secretion. The protocol used was previously described by Inglis et al. (1996). Briefly, upper lung lobes were removed from humanely killed pigs and the bronchi dissected free. Side branches were ligated and the bronchi were placed in a bath containing Krebs Ringer Bicarbonate (KRB). After warming to 37°C, bronchi were cannulated and perfused with fresh KRB. Transepithelial PD was measured using a glass microelectrode inserted into the lumen and a reference electrode connected to the bath via a salt bridge. Basolateral addition of 50µM ACh resulted in hyperpolarisation of the PD by 1.62 ± 0.17mV (mean ± SEM) n=33. 15 minute pre-treatment with 10µM PZ resulted in significant inhibition (46.51 ± 16.77% n=8 P=0.03) of this response (Students paired t-test used throughout, significance P<0.05). 1µM PZ pre-treatment resulted in an increased response to ACh (41.91 ± 31.62% n=8) although not significant (P=0.80). The same experiment has been performed using 1µM, 100nM and 10nM DAMP resulting in inhibition of the response to ACh by 87.44 ± 10.84% (P=0.02 n=6), 85.92 ± 2.35% (P=0.00 n=5) and 8.80 ± 9.05% (P=0.20 n=5) respectively. The IC₅₀ values of inhibition by 4-DAMP and Pirenzipine are 2.2 x10^-8M and 7.9 x10^-6M respectively. In summary, ACh addition results in hyperpolarisation of the transepithelial PD consistent with increased anion secretion into the bronchial lumen. The data presented here suggest that this ACh induced increase in PD is mediated largely by the M3 subtype of muscarinic receptors.