AbstractPeriportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar heterogeneity of amino acid transport activities related to glutamine and ammonia metabolism. Immunocytochemical staining of the respective subpopulations for glutamine synthetase demonstrated that periportal subpopulations were essentially free of glutamine synthetase‐positive cells, whereas perivenous subpopulations showed a 2‐ to 3‐ fold enrichment of glutamine synthetase‐positive hepatocytes. The high perivenous/periportal ratio of 59 found for glutamine synthetase activity as well as the perivenous/periportal ratios of other marker enzymes further indicated the good separation of periportal and perivenous cells.α‐Aminoisobutyric acid, histidine and glutamate were used to determine the distribution pattern of amino acid transport systems A, N and G−, as well as of the sodium‐independent uptake of these compounds 1 hr after isolation and after maximal hormonal stimulation during primary culture. The strong heterogeneity of the sodium‐independent transport of histidine, characterized by higher perivenous transport rates [perivenous/periportal ratio: 1.5 (1 hr) to 3.5 (48 hr)], suggests a significant role of facilitated diffusion, presumably in glutamine export. Conversely, the strong heterogeneity of the sodium‐dependent glutamate transport (System G−) characterized by higher uptake rates in nonstimulated [perivenous/periportal ratio: 6.6 (1 hr)] and in hormonally treated perivenous hepatocytes (perivenous/periportal ratio: 2.2) reflects its possible significance with respect to the substrate availability for glutamine synthesis. The observed heterogeneities provide a basis for understanding how substrate fluxes related to glutamine metabolism might be established and regulated. In addition, our findings indicate that periportal and perivenous cells retain their zonal characteristics with respect to amino acid transport in primary cultur