The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.