A method has been devised to quantitate rates of ketogenesis (acetoacetate + β-hydroxybutyrate production) in discrete regions of the liver lobule based on changes in NADH fluorescence. In perfused livers from fasted rats, ketogenesis was inhibited nearly completely with either 2-bromooctanoate (600 μm) or 2-tetradecylglycidic acid (25 μm). During inhibition of ketogenesis, a linear relationship (r = 0.90) was observed between decreases in NADH fluorescence detected from the liver surface and decreases in ketone body production. NADH fluorescence was monitored subsequently from individual regions of the liver lobule by placing microlight guides on periportal and pericentral regions of the liver lobule visible on the liver surface. Rates of ketogenesis in sublobular regions were calculated from regional decreases in NADH fluorescence and changes in the rate of ketone body formation by the whole liver during infusion of inhibitors. In the presence of bromooctanoate, ketogenesis was reduced 80% and local rates of ketogenesis were decreased 31 ± 4 μmol/g/h in periportal areas and 28 ± 3μmol/g/h in pericentral regions. Similar results were observed with tetradecylglycidic acid. Therefore, it was concluded that submaximal rates of ketogenesis from endogenous, mainly long-chain fatty acids are nearly equal in periportal and pericentral regions of the liver lobule in livers from fasted rats. Rates of ketogenesis and NADH fluorescence were strongly correlated during fatty acid infusion. Infusion of 250 μm oleate increased NADH fluorescence maximally by 8 ± 1% over basal values in periportal regions and 17 ± 4% in pericentral areas. Local rates of ketogenesis, calculated from these changes in fluorescence, increased 35 ± 6 μmol/g/h in periportal areas and 55 ± 5 μmol/g/h in pericentral regions. Thus, oleate stimulated ketogenesis nearly 60% more in pericentral than in periportal regions of the liver lobule.