The role of plasma glucose in the regulation of lipid metabolism in humans, independent of associated changes in hormone concentrations, is controversial. Therefore we examined the role of glucose in the regulation of lipolysis and free fatty acid (FFA) reesterification in six healthy lean male volunteers. Blood glucose concentration was clamped at either 5 or 10 mM during 2-h pancreatic-pituitary clamps. Glycerol and palmitate turnover were measured by isotope dilution ([1-14C]palmitate and [2H5]glycerol). All hormone concentrations were the same during the euglycemic and hyperglycemic studies. FFA turnover, which represents the difference between lipolysis and FFA reesterification, was reduced 30% by hyperglycemia (29 ± 2 vs. 20 ± 3 μmol·kg fat mass-1·min-1, P < 0.05). Glycerol turnover, which represents lipolysis only, was reduced to a similar extent (9.4 ± 0.9 vs. 6.2 ± 0.7 μmol·kg fat mass-1·min-1, P < 0.05). We conclude that glucose regulates lipid metabolism, independently of changes in hormone concentrations. The equivalent suppression of glycerol and FFA turnover indicates that the effect is mediated by suppression of lipolysis and not by stimulation of FFA reesterification.