Abstract New methods have been developed to monitor metabolic events non-invasively within periportal and pericentral regions of perfused rat livers. These techniques utilizes two-fiber micro-light guides and miniature oxygen electrodes positioned on identified lobular regions of the perfused liver based on differential pigmentation of periportal and pericentral areas. Two-fiber micro-light guides detect the fluorescence of native and introduced fluors and are used to monitor redox changes of endogenous pyridine nucleotides and the generation of fluorescent products (e.g., 7-hydroxycoumarin) from exogenous substrates. Changes in fluorescence detected with two-fiber micro-light guides are correlated with changes measured with large, multi-fiber light guides and with whole organ rates of metabolism. Subsequently, local rates are estimated. With these techniques, we show that (a) rates of ethanol and acetaldehyde metabolism are similar in periportal and pericentral regions of the liver lobule; (b) mixed-function oxidation predominantes in pericentral regions in livers from phenobarbital-treated rats; (c) rates of sulfation of 7-hydroxycoumarin are greater in periportal than in pericentral hepatocytes; and (d) oxygen uptake is approximately 3-fols greater in periportal than in pericentral areas of the liver lobule.