The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca2+ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca2+]i, in perfused rat pancreatic ducts using the Ca(2+)-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, Vbl, of individual cells. The resting basal [Ca2+]i was relatively high, corresponding to 263 +/- 28 nmol/l, and it decreased rapidly to 106 +/- 28 nmol/l after removal of Ca2+ from the bathing medium (n = 31). Carbachol increased [Ca2+]i in a concentration-dependent manner. At 10 mumol/l the fura-2 fluorescence ratio increased by 0.49 +/- 0.06 (n = 24), corresponding to an increase in [Ca2+]i by 111 +/- 15 nmol/l (n = 17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67 +/- 0.06 and 1.01 +/- 14 (n = 46; 12), corresponding to a [Ca2+]i increase of 136 +/- 22 nmol/l and 294 +/- 73 nmol/l respectively (n = 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized Vbl from -62 +/- 3 mV to -70 +/- 3 mV, an effect which was in some cases only transient (n = 7). This effect of ATP was different from that of carbachol, which depolarized Vbl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of Vbl (n = 4). Several other putative agonists of pancreatic HCO3- secretion were also tested for their effects on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)