1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture. 2. Ductal fluid secretion was measured using micropuncture techniques. Intracellular free calcium ([Ca2+]i) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion. 3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0.4 microM and maximal secretion was achieved with 10 microM ACh. These effects of ACh were blocked by atropine and by removal of extracellular Ca2+. 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar. 5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]i. 6. The [Ca2+]i response evoked by ACh resulted from both mobilization of intracellular Ca2+ stores and influx of Ca2+ from the extracellular space. 7. The Ca2+ ionophore, ionomycin, mimicked the effect of ACh on ductal [Ca2+]i and fluid secretion. 8. We conclude that ACh stimulates fluid secretion from rat pancreatic duct cells by activating a 'Ca2+ pathway' which is distinct from the well documented 'cyclic AMP pathway' utilized by secretin.