Rates of oxygen uptake in periportal and pericentral regions of the hemoglobin-free perfused rat liver were measured successfully for the first time with a miniature oxygen electrode (tip diameter, 50–60 microns) by stopping the flow of perfusate and measuring the rate of decrease of oxygen concentration on the surface of the liver. Rates of oxygen uptake in periportal and pericentral areas were 131 +/- 9 (mean +/- SD) and 56 +/- 14 mumol X g-1 X h-1 in livers from fed, phenobarbital-treated rats, respectively. In livers from fasted rats, rates of O2 uptake of 141 +/- 12 and 89 +/- 11 mumol X g-1 X h-1 were observed in periportal and pericentral regions, respectively. Similar data were obtained in livers from normal rats. Thus, periportal hepatocytes have higher rates of oxygen uptake than pericentral cells in both the fed and fasted states. Rates of oxygen uptake were not affected in periportal regions by fasting. In contrast, rates of oxygen uptake by hepatocytes in pericentral areas were significantly (P less than 0.001) greater in livers from fasted than from fed rats. This significant increase in oxygen uptake in pericentral hepatocytes as a result of fasting is consistent with the hypothesis that glycolysis occurs predominantly in pericentral regions of the liver lobule.