To determine the impact of variable plasma insulin concentrations and glycemia on the partitioning of whole body glucose metabolism between glycolysis and glucose storage, we estimated endogenous hepatic glucose production and rates of in vivo glycolytic flux (GF) and glucose storage (GS) in six normal dogs from the generation of plasma tritiated water (3H2O) and [3-3H]glucose specific activity during 150 min of somatostatin euglycemic (E) and hyperglycemic (H) clamps at hypoinsulinemic, basal, intermediate, and high insulin levels. During both E and H clamps, overall rates of GF and GS increased with the rising insulin levels, but the relative contributions to in vivo glucose disposal of GF decreased, whereas GS rose progressively with increasing insulin levels. The relative contribution of GS during H to overall glucose disposal was greater at the lower insulin level. In addition, in absolute terms, GF and GS were significantly higher (P < 0.05) during H than during E at all insulin levels. Moreover, the incremental rise in GF induced by H was equal for the low to intermediate insulin levels tested, independent of the prevailing free fatty acid (FFA) levels. However, when whole body glucose disposal rates were matched, GF and GS rates were independent of the coexisting glycemia, insulin, and/or FFA levels. We conclude that 1) insulin has a major impact on the intracellular fate of infused glucose, with a lesser but significant effect of hyperglycemia per se on these processes; 2) the magnitude of the hyperglycemia-induced increase in GF is independent of the prevailing insulin level from low to intermediate levels; and 3) in vivo GF and GS are dependent on the net rate of glucose uptake into cells but independent of absolute FFA levels or whether glucose uptake is stimulated by raised insulin or glucose levels.