Nitric oxide (NO) and guanosine 3′,5′-cyclic monophosphate (cGMP) have recently been shown to stimulate bile acid–independent bile flow in the isolated perfused rat liver (IPRL). However, the cellular origin and mechanisms of this choleresis have not yet been determined. To address these questions, we examined the effects of NO and cGMP on bile secretion in isolated rat hepatocyte couplets (IRHC) and in isolated bile duct units (IBDU), both of which are isolated cell systems in which cell polarity is maintained and secretion can be measured directly. Changes in the area of the canalicular and ductular lumens were determined in IRHC and IBDU, respectively, as indicators of the rate of fluid secretion using video microscopy. In addition, Cl/HCO3 exchanger activity in IBDU was evaluated by measuring changes in intracellular pH (pHi ) after Cl removal/readmission by microfluorometric methods. In the presence of HCO3, both the NO donor, S-nitroso-acetyl-penicillamine (SNAP), and the cell-permeant cGMP analogue, dibutyryl cGMP (DBcGMP), stimulated canalicular bile secretion (P < .05), as did the cell-permeant cAMP analogue, dibutyryl cAMP (DBcAMP) (P < .05). Removal of HCO3 from the buffer completely abolished the choleretic effects of DBcGMP, but had no effect on NO-induced choleresis. In contrast, secretion in IBDU was not stimulated following incubations with SNAP or DBcGMP over 30 minutes, whereas DBcAMP and secretin, a cholangiocyte secretagogue and cAMP agonist, both had a marked effect on ductular secretion over this same time interval (P < .05). SNAP also had no effect on Cl/HCO3 exchanger activity in IBDU, and inhibition of endogenous NO synthesis by NG-monomethyl-l -arginine (l -NMMA) did not alter secretin-induced stimulation of ductular bile secretion and Cl/HCO3 exchanger activity. In summary, NO and cGMP stimulate bile secretion exclusively at the the level of hepatocytes, whereas cAMP mediates choleresis at both hepatocyte and bile duct levels. These findings may have important implications for the regulation of ductular bile secretion by hormones and neuropeptides, as well as under pathological conditions with increased hepatic NO synthesis.