Many enzymes are distributed heterogeneously within the liver lobule. The factors that play a determining role in the establishment and maintenance of these heterogeneous expression patterns have not yet been identified. To investigate whether the composition of the afferent hepatic blood plays a crucial role in the maintenance of the heterogeneity of gene expression of the parenchymal cells within the liver lobule, we changed the source of the afferent hepatic blood by microsurgical techniques. Three different groups of experimental animals were studied: rats with livers that are perfused with portal blood only (ligation of the hepatic artery), with caval blood only (portocaval transposition and ligation of the hepatic artery) and arterial blood only (portocaval shunt, arterialization of the distal end of the portal vein and ligation of the hepatic artery). To study differences in gene expression patterns, we chose enzymes that have a heterogeneous expression pattern within the liver lobule: the periportally located enzymes carbamoylphosphate synthase, succinate dehydrogenase, phosphoenolpyruvate carboxylkinase and the pericentrally located enzymes glutamine synthase, glutamate dehydrogenase and NADPH–cytochrome P-450 reductase. To eliminate the potential interference of the long half-lives of some of these proteins on the interpretation of the results, we also studied the distribution of the mRNAs of carbamoylphosphate synthase, glutamine synthase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase. The animals were studied 2 wk after the operations. On the basis of their changes in body weight the animals were in steady state for at least a week. The patterns of gene expression of the enzymes studied did not change, regardless of the source of the altered afferent hepatic blood. The changes in gene expression that were observed in animals that did not regain their preoperative weight were shown to be caused by a limited intake of food. This study demonstrates that the physiological position of the liver within the circulation (i.e., between the gastrointestinal tract and the systemic circulation) is not as critical as is often stated and is certainly not essential for the maintenance of liver cell heterogeneity. The data suggest that the direction of the bloodstream (i.e., the existence of an upstream and a downstream compartment) is a major determinant of zonation of gene expression. (HEPATOLOGY 1993;18:1144-1153).