Active secretion of electrolytes by hepatocytes is believed to be responsible for bile acid-independent canalicular bile flow (BAICF). Theophylline, which enhances BAICF, has been shown to enhance electrogenic Cl- secretion in a number of other epithelia. Such transport is dependent on Na+ and Cl-. Thus, the mechanism of theophylline choleresis may also involve stimulation of electrogenic Cl- secretion of the liver. This hypothesis was tested by studying the effect of ion substitution on theophylline choleresis in isolated perfused rat livers. Addition of theophylline (0.1 mmol) and dibutyryl cAMP (0.05 mmol) to 100 ml perfusate, in a single dose, increased bile flow and biliary secretion of Na+ and Cl- reversibly. These effects of theophylline were virtually abolished when perfusate Na+ (146 mM) was replaced by Li+ (146 mM) or choline+ (120 mM), and when Cl- (127 mM) was replaced by 120 mM NO-3, acetate- or isethionate-. Since even the permeable ions like Li+ and NO-3 could not substitute for Na+ and Cl-, these results show that the effect of theophylline on BAICF is specifically dependent on the presence of Na+ and Cl- in the perfusate. We propose, by analogy to other epithelia, that an electrogenic Cl- secretion mechanism is present in the liver. Theophylline, acting via cAMP, stimulates this transport process, thereby enhancing BAICF.