A high-sucrose diet (SU) decreases insulin action in the liver (Pagliassotti MJ, Shahrokhi KA, and Moscarello M. Am J Physiol Regulatory Integrative Comp Physiol 266: R1637-R1644, 1994). The present study was conducted to characterize the effect of SU on glucagon action in isolated periportal (PP) and perivenous (PV) hepatocytes by measuring glucagon-stimulated glycogenolysis and glucose release. Male rats were fed a SU (68% sucrose) or starch diet (ST, 68% starch) for 1 wk, and hepatocytes were isolated from PP or PV regions (n = 4/diet/cell population). Hepatocytes were incubated for 1 h in the presence of varying concentrations of glucagon (0-100 nM). In PP and PV cells, glucagon stimulation of glucose release and glycogenolysis (sum of glucose release and lactate accumulation) was not significantly different between SU and ST cells. However, in the SU PP cells, glucose release was increased compared with ST PP cells, both in the absence of glucagon (76.1 ± 4 vs. 54.8 ± 3 nmol · h¹ · mg cell wet wt¹) and at all glucagon concentrations. In SU-fed PV cells, glucose release was increased compared with ST PV cells in the absence of glucagon (79.3 ± 5 vs. 56.4 ± 5 nmol · h¹ · mg cell wet wt¹) and at low glucagon concentrations. Maximal glucose-6-phosphatase activity (in nmol · min¹ · mg protein¹) was elevated in SU compared with ST cells (61.4 ± 3 vs. 37.5 ± 4 in PP and 37.5 ± 4 vs. 29.5 ± 3 in PV cells). In contrast, maximal glucokinase activity (in nmol · min¹ · mg protein¹) was elevated in ST compared with SU cells (15.9 ± 2 vs. 12.1 ± 1 in PP and 19.4 ± 2 vs. 14.2 ± 1 in PV cells). These data demonstrate that SU increases the capacity for glucose release in both PP and PV hepatocytes, in part because of reciprocal changes in glucose-6-phosphatase and glucokinase.