It has been established, mainly by histochemical and immunohistochemical studies, that liver cells are functionally heterogeneous, with periportal hepatocytes (PPHs) being predominantly gluconeogenic and perivenous hepatocytes (PVHs) being glycolytic. We therefore investigated the region-specific functional effects of glucagon on glycogenolysis and gluconeogenesis in isolated PPHs and PVHs prepared by the digitonin-collagenase method. BB rats, a model of insulin-dependent diabetes, were used to study the region-specific heterogeneity of gluconeogenesis in the diabetic state. Although glycogen content was not different between PVHs and PPHs in rats fed the normal diet, basal glucose release was 1.37 times greater in PVHs than in PPHs (P <.05). The increase in glucose release stimulated by 0.01 to 0.1 nmol/L glucagon was 1.52 times greater in PVHs than in PPHs (P < .05), whereas no differences were seen in response to 1 to 100 nmol/L glucagon. Glucose release from gluconeogenic substrates was 1.57 times greater in the PPHs than in the PVHs of fasted normal rats (P < .05), whereas the increase in gluconeogenesis produced by glucagon was not different between PPHs and PVHs. The glucagon-binding capacity, the cAMP release, and the increase in intracellular Ca2+ stimulated by glucagon were not different between PPHs and PVHs in the fed or fasted states. Gluconeogenesis from gluconeogenic substrates was 1.52 times greater in the PPHs than in the PVHs of fasted nondiabetic BB rats (P < .05). After the development of diabetes, the gluconeogenic capacity in PVHs increased to the level observed in PPHs, but that in PPHs did not change. Thus there was no difference in gluconeogenesis between the PPHs and PVHs of diabetic BB rats. In both the PPHs and PVHs of diabetic BB rats, the 0.01 to 100 nmol/L glucagon-induced increase in gluconeogenesis was greater than that in PPHs from nondiabetic BB rats (2.30 and 3.07 times, P < .01, respectively). We conclude that PPHs and PVHs of normal rat liver express region-specific differences in their glycogenolytic and gluconeogenic responses to glucagon. In diabetic BB rats, the difference in the gluconeogenic capacity between PPHs and PVHs disappeared, whereas glucagon-induced gluconeogenesis was enhanced.