In livers of fed rats and in perfused livers supplied with a physiological portal glutamine concentration of 0.6 mM, the mitochondrial and cytosolic glutamine concentrations are 20 mM and 7 mM, respectively, thus, the mitochondrial/cytosolic glutamine concentration gradient is 2-3. Uptake and release of glutamine by periportal and perivenous hepatocytes occurs predominantly by an Na+-dependent transport system (so-called system 'N'). Histidine in near-physiological concentrations inhibits both glutamine uptake by periportal hepatocytes and its release by perivenous hepatocytes. This is not due to an inhibition of glutamine-metabolizing enzymes by histidine or its metabolites. With physiological portal glutamine concentrations (0.6 mM), stimulation of glutaminase flux or of glutamine transaminase flux is followed by a decrease of hepatic glutamine levels to about 80% or 30%, respectively, glutamine levels are further decreased to 50% or 20% in the presence of histidine. When glutamine is synthesized endogenously (no glutamine added), the histidine-induced inhibition of glutamine release is paralleled by a 210% increase of the hepatic tissue level of glutamine. In experiments with and without methionine sulfoximine and in the absence of added glutamine, the glutamine content in the small perivenous hepatocyte population containing glutamine synthetase is estimated to be about 3.5 mumol/g wet weight and that in the periportal hepatocytes as low as 0.1 mumol/g wet weight. In contrast to the prevailing view, it is concluded that glutamine transport across the plasma membrane of hepatocytes is a potential regulatory site in glutamine degradation and synthesis, especially under the influence of effectors like histidine.