While intrahepatic bile duct epithelial cells secrete bile through transport of ions and water, the physiological mechanisms regulating ductular bile secretion are obscure, in part because of the lack of suitable experimental models. We report here the successful micropuncture of the lumen of isolated intrahepatic bile ducts and direct measurements of ductular ion secretion. Intact, polarized bile duct units (BDUs) were isolated from livers of normal rats by enzymatic digestion and microdissection. BDUs were cultured and mounted on a microscope in bicarbonate-containing buffer, and the lumens were microinjected with 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF)-dextran. Lumenal pH was measured by ratio imaging of BCECF fluorescence using digitized video fluorescent microscopy. After 36 hr in culture, the ends of BDUs sealed, forming closed compartments. After lumenal microinjection of BCECF-dextran, fluorescence was stable at the pH-insensitive wavelength, indicating no dye leakage. Serial changes in pH of extralumenal buffers containing pH-gradient collapsing ionophores allowed us to establish reliable standard curves relating fluorescence ratio to lumenal pH (r = 0.99; P < 0.001). By this approach, the basal pH inside the lumen of BDUs was 7.87 +/- 0.08 units (n = 9), 0.47 unit higher (P < 0.001) than the bathing buffer pH. Addition of 100 microM forskolin increased (P = 0.02) the lumenal pH from 7.78 +/- 0.06 to 7.97 +/- 0.06 units (n = 5); the forskolin effect was completely abolished by incubation of BDUs in HCO3-/CO2-free buffer. Moreover, forskolin caused a 50-fold increase in cAMP levels in BDUs. The observations are consistent with cAMP-dependent, active lumenal HCO3- secretion by BDUs. Furthermore, they demonstrate the suitability of the BDU model for studying regulatory and mechanistic aspects of ductular bile secretion.