The redox gradient along the sinusoid in the rat liver was studied using a redox scanner, a device based on tissue fluorescence scanning spectroscopy measuring the fluorescence signals of oxidized flavoprotein (FP) and reduced pyridine nucleotide (PN). The FP/(FP + PN) ratio reflects the mitochondrial redox state in the liver tissue. The distribution of mitochondrial redox state on the scanned area is expressed as two-dimensional gray-scale images with a 20 μm resolution. Using this instrument, we have scanned a 2.5 × 2.5 mm area of the frozen rat liver sample to investigate the redox gradient within acini and the effects of glucagon on the changes in the redox distribution. The redox images obtained in the perfused livers showed mosaic patterns implicating a regular heterogeneity of redox state in an acinus. The analysis of gradient curve, furthermore, clarified that the redox level in an acinus decreased sigmoidally from the periportal to the pericentral region. Glucagon, which has been reported to reduce the intracellular redox state, decreased the redox potential in whole acini, especially, in the periportal region, when compared with the perfusion without glucagon. These results strongly indicate an intraacinus heterogeneity of glucagon function, with glucagon selectively operating in the upstream of the sinusoid.