To investigate intercellular compartmentation of liver metabolism, we have recently introduced new procedures for quantitative assessment of metabolic liver cell heterogeneity both along sinusoids of portal and septal origins as well as at the level of the parenchymal unit, and also for three-dimensional imaging of enzyme and metabolite distribution. As part of the evaluation of the role of metabolic liver cell heterogeneity for the regulation of net substrate flux in the glucose-6-phosphatase/glucokinase system, and for the reduction of glucose/glucose-6-P recycling through spatial separation of these antagonistic enzymes, these techniques were used on livers from male rats. They served to obtain distribution data on glucose-6-phosphatase (the hydrolytic component of the glucose-6-phosphatase/glucokinase system) and its substrate, glucose-6-P, during the postresorptive phase (i.e., a metabolic state of net glucose release). Glucose-6-phosphatase (Vmax) and glucose-6-P were shown to decrease along the sinusoidal axis, and values of both parameters were significantly higher along sinusoids of portal than septal origin. Distribution of in vivo rates of glucose-6-P hydrolysis indicates the importance of metabolite distribution for in vivo regulation of liver cell function, insofar as it considerably increases the degree of heterogeneity among hepatocytes over that of maximal rates of glucose formation. Histo- and microchemical data support the concept of a “lobular parenchymal unit” composed of “primary lobules,” and justify the conclusion that hepatocyte function, in addition to the hormonal and nutritional states of the animal, not only depends upon cell location along the sinusoidal axis, but also on the origin of sinusoids.