Bile formation by the liver is largely dependent on the transport of bile acids by hepatocytes. This process is thought to result from Na-coupled uptake of bile acids into the cell and voltage-dependent, carrier-mediated transport from cell to canaliculus. However, the dependence of bile secretion on membrane potential has not yet been observed. In this study, the effect of changes in membrane potential differences on bile secretion was tested by impaling rat hepatocyte couplets with microelectrodes, changing membrane potential by intracellular current injection, and measuring fluid secretion by optically determining canalicular size. In the presence of 50 microM taurocholate, membrane potential was -33.3 +/- 5.8 mV and canalicular area increased by 6 +/- 6%/min, corresponding to a fluid secretion rate of 2-4 fl/min. In contrast, when intracellular voltage was suddenly changed to -109.9 +/- 15.0 mV, the canalicular area increased by 20 +/- 4%/min, corresponding to a secretion rate of 19 fl/min. When these experiments were repeated in the absence of taurocholate, the negative clamp had no effect on canalicular size. Taurocholate itself did not alter membrane potential. These findings support the hypothesis that canalicular bile secretion depends on a process equivalent to electrodiffusion. We therefore conclude that membrane voltage is a driving force for taurocholate-dependent fluid secretion by the liver.