Aims/hypothesis. Non-esterified fatty acids and glycerol could stimulate gluconeogenesis and also contribute to regulating hepatic glycogen stores. We examined their effect on liver glycogen breakdown in humans.¶Methods. After an overnight fast healthy subjects participated in three protocols with lipid/heparin (plasma non-esterified fatty acids: 2.2 ± 0.1 mol/l; plasma glycerol: 0.5 ± 0.03 mol/l; n = 7), glycerol (0.4 ± 0.1 mol/l; 1.5 ± 0.2 mol/l; n = 5) and saline infusion (control; 0.5 ± 0.1 mol/l; 0.2 ± 0.02 mol/l; n = 7). Net rates of glycogen breakdown were calculated from the decrease of liver glycogen within 9 h using ¹³C nuclear magnetic resonance spectroscopy. Endogenous glucose production was measured with infusion of D-[6,6-²H₂]glucose.¶Results. Endogenous glucose production decreased by about 25 % during lipid and saline infusion (p < 0.005) but not during glycerol infusion (p < 0.001 vs lipid, saline). An increase of plasma non-esterified fatty acids or glycerol reduced the net glycogen breakdown by about 84 % to 0.6 ± 0.3 μmol · kg^–1· min^–1 (p < 0.001 vs saline: 3.7 ± 0.5 μmol · kg^–1· min^–1) and by about 46 % to 2.0 ± 0.4 μmol · kg^–1· min^–1 (p < 0.01 vs saline and lipid), respectively. Rates of gluconeogenesis increased to 11.5 ± 0.8 μmol · kg^–1· min^–1 (p < 0.01) and 12.8 ± 1.0 μmol · kg^–1· min^–1 (p < 0.01 vs saline: 8.2 ± 0.7 μmol · l^–1· min^–1), respectively.¶Conclusion/interpretation: An increase of non-esterified fatty acid leads to a pronounced inhibition of net hepatic glycogen breakdown and increases gluconeogenesis whereas glucose production does not differ from the control condition. We suggest that this effect is not due to increased availability of glycerol alone but rather to lipid-dependent control of hepatic glycogen stores. [Diabetologia (2001) 44: 48–54]