Spatial and temporal alterations in carbon tetrachloride-induced oxidative stress were studied in perfused hepatic microcirculatory units of the rat by digital imaging microscopic fluorography using dichlorofluorescin diacetate, a hydroperoxide-sensitive fluorogenic probe. The surface of the liver loaded with dichlorofluorescin was microscopically visualized, and the fluorescence of dichlorofluorescin (DCF), a highly fluorescent molecule generated by hydroperoxide-mediated oxidation of dichlorofluorescin, was digitally processed. After completing the experiments, fluorescein-labeled albumin was injected into the perfusate to confirm the state of sinusoidal perfusion and the topography of hepatic microangioarchitecture in the area studied. During transportal infusion of carbon tetrachloride, DCF fluorescence was observed predominantly in the pericentral area and was attenuated by pretreatment with SKF-525A, suggesting the involvement of cytochrome P-450. After peaking at the maximum level, the pericentral DCF fluorescence gradually decreased in parallel to the loss of viability, implicating the causal role of intracellular hydroperoxide formation in hepatocellular injury. In retrogradely perfused liver, in which intralobular O2 gradient was reversed, no significant activation of DCF fluorescence was observed among hepatic lobules. It is therefore conceivable that the zonal heterogeneity of carbon tetrachloride-induced lipid peroxidation may depend on at least two factors, sinusoidal oxygenation and the intralobular distribution of cytochrome P-450. Furthermore, development of the current technique will provide a useful method to investigate the microtopography of oxidative stress in organ microcirculatory units.