Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO₃^- requirement for secretin-evoked secretion in mouse, rat and guinea-pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nM secretin and 5 µM forskolin in the absence of extracellular HCO₃^-. In guinea-pig ducts, however, fluid secretion was totally dependent on HCO₃^-. Forskolin-stimulated fluid secretion by mouse and rat ducts in the absence of HCO₃^- was dependent on extracellular Cl^- and was completely inhibited by bumetanide (30 µM). It was therefore probably mediated by a basolateral Na⁺-K⁺-2Cl^- cotransporter. In the presence of HCO₃^-, forskolin-stimulated fluid secretion was reduced 40% by bumetanide, 50% by inhibitors of basolateral HCO₃^- uptake (3 µM EIPA and 500 µM H₂DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin-evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na⁺/H⁺ exchange and Na⁺-HCO₃^- cotransport mediating HCO₃^- uptake, and Na⁺-K⁺-2Cl^- cotransport mediating Cl^- uptake. The absence or inactivity of the Cl^- uptake pathway in the guinea-pig pancreatic ducts may help to account for the much higher concentrations of HCO₃^- secreted in this species.