The present study sought to determine whether elevated plasma free fatty acids (FFAs) alter the ability of insulin and glucose to regulate splanchnic as well as muscle glucose metabolism. To do so, FFAs were increased in 10 subjects to approximately 1 mmol/l by an 8-h Intralipid/heparin (IL/Hep) infusion, whereas they fell to levels near the detection limit of the assay (<0.05 mmol/l) in 13 other subjects who were infused with glycerol alone at rates sufficient to either match (n = 5, low glycerol) or double (n = 8, high glycerol) the plasma glycerol concentrations observed during the IL/Hep infusion. Glucose was clamped at approximately 8.3 mmol/l, and insulin was increased to approximately 300 pmol/l to stimulate both muscle and hepatic glucose uptake. Insulin secretion was inhibited with somatostatin. Leg and splanchnic glucose metabolism were assessed using a combined catheter and tracer dilution approach. Leg glucose uptake (21.7 +/- 3.5 vs. 48.3 +/- 9.3 and 57.8 +/- 11.7 micromol x kg(-1) leg x min(-1)) was lower (P < 0.001) during IL/Hep than the low- or high-glycerol infusions, confirming that elevated FFAs caused insulin resistance in muscle. IL/Hep did not alter splanchnic glucose uptake or the contribution of the extracellular direct pathway to UDP-glucose flux. On the other hand, total UDP-glucose flux (13.2 +/- 1.7 and 12.5 +/- 1.0 vs. 8.1 +/- 0.5 micromol x kg(-1) x min(-1)) and flux via the indirect intracellular pathway (8.4 +/- 1.2 and 8.1 +/- 0.6 vs. 4.8 +/- 0.05 micromol x kg(-1) x min(-1)) were greater (P < 0.05) during both the IL/Hep and high-glycerol infusions than the low-glycerol infusion. In contrast, only IL/Hep increased (P < 0.05) splanchnic glucose production, indicating that elevated FFAs impaired the ability of the liver to autoregulate. Splanchnic insulin extraction, directly measured using the arterial and hepatic vein catheters, did not differ (67 +/- 3 vs. 71 +/- 5 vs. 69 +/- 1%) during IL/Hep and high- and low-glycerol infusions. We conclude that elevated FFAs exert multiple effects on glucose metabolism. They inhibit insulin- and glucose-induced stimulation of muscle glucose uptake and suppression of splanchnic glucose production. They increase the contribution of the indirect pathway to glycogen synthesis and impair hepatic autoregulation. On the other hand, they do not alter either splanchnic glucose uptake or splanchnic insulin extraction in nondiabetic humans.