The aim of these studies was to investigate the effect of hyperglycemia with or without hyperinsulinemia on hepatic gluconeogenic flux, with the hypothesis that inhibition would be greatest with combined hyperglycemia/hyperinsulinemia. A glycogen phosphorylase inhibitor (BAY R3401) was used to inhibit glycogen breakdown in the conscious overnight fasted dog and the effects of a two fold rise in plasma glucose level (HI group) accompanied by 1) euinsulinemia (HG group) or 2) a four fold rise in plasma insulin were assessed over a five hour experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. In the HG group net hepatic glucose uptake and net hepatic lactate output substantially increased. There was little or no effect on the net hepatic uptake of gluconeogenic precursors other than lactate (amino acids and glycerol) nor on the net hepatic uptake of free fatty acids compared to the control group. Consequently, while hyperglycemia had little effect on gluconeogenic flux to G6P, net hepatic gluconeogenic flux was reduced, due to increased hepatic glycolytic flux during hyperglycemia. Net hepatic glycogen synthesis was increased by hyperglycemia. The effect of hyperglycemia on gluconeogenic flux to G6P and net hepatic gluconeogenic flux was similar. We conclude that in the absence of appreciable glycogen breakdown the increase in glycolytic flux that accompanies hyperglycemia results in decreased net carbon flux to G6P, but had no effect on gluconeogenic flux to G6P.