Acetyl-CoA carboxylase (ACC) activity in the liver varies markedly as a function of the nutritional state and is subject to complex regulation involving variable enzyme content, enzyme specific activity due to variable phosphorylation, and zonation within the hepatic lobule. A 5'-AMP-activated protein kinase (AMPK) has been identified as the major regulatory kinase active on ACC. Employing dual-digitonin pulse perfusion, the effect of varying nutrition on periportal and perivenous zonation of ACC and AMPK activity within the liver has been characterized. During the transition from fasting to refeeding with high-carbohydrate chow, total ACC activity is increased 11- to 17-fold. This induction of total ACC activity is accounted for by a 4.5- to 6-fold increase in the content of the two major ACC isoforms and by a 2.5-to 3-fold increase in enzyme specific activity (units per mg ACC). Despite a small perivenous preponderance of ACC protein, a gradient of activity to the periportal side, due to this increase in specific activity, is observed in fasted rats and during early refeeding. After 24-48 h of refeeding, maximal induction of both ACC protein and specific activity is observed with obliteration of this total activity gradient. 5'-AMP-activated protein kinase activity is maximal in the fasted rat and is zonated to the perivenous side. During refeeding, this activity is rapidly markedly diminished with abolition of this gradient, correlating inversely with the activation of ACC over a narrow range of kinase activity. Activities of casein kinase II, myelin basic protein kinase(s), and ribosomal S6 kinase(s) show no zonation. These data suggest that the zonal activity of the 5'-AMP-activated protein kinase is responsible, in part, for the intrahepatic gradient in ACC activity and that the regulation of this kinase is responsible for the variations in ACC-specific activity that occur during varying nutrition.