To identify a role for amino acids in cholestasis associated with total parenteral nutrition, we measured bile formation by the isolated perfused rat liver in the presence and absence of added amino acids. All livers were infused,constantly with sodium [14C]taurocholate (0.28 μmoles per min) for 90 min. At 40 min, a primed-constant infusion of a synthetic L-amino acid mixture (121 + 19.3 μmoles of N per min) was administered for an additional 50 min. Mean bile flow rates during the amino acid infusion were reduced from 15.4 μl per min per 10 gm liver weight to 10.4 μl per min per 10 gm (p < 0.005). There was no significant change during saline infusion of control livers, and there was no significant difference in perfusate osmolalities in the two groups. Although biliary recovery of infused taurocholate was slightly lower in the experimental perfusions than in controls (95.3% vs. 101.7%, p < 0.05), there was no significant reduction in taurocholate excretion rate during the infusion in either group. Bile flow changes were related to ambient concentrations and net fluxes of individual amino acids in the perfusate. Of the 14 infused amino acids, glycine and arginine achieved levels >3 times greater than reported physiological postprandial portal venous concentrations in the rat, and together constituted about 25% of the 9 0-min perfusate amino acids (8.3 mM). The highest net hepatic uptake was for glycine (125 μmoles per hr per 10 gm), which was almost 50% of its infusion rate. These observations lead us to propose that nonphysiological hepatic concentrations and uptake of certain amino acids interfere with bile formation, particularly with bile acid-independent flow.