This study evaluated lactate disposal via gluconeogenesis as well as effects of FFA availability on gluconeogenesis via pyruvate (GNG(PYR)) in patients with mitochondrial myopathy due to complex I deficiency (CID). The rates of GNG(PYR) were measured in three CID patients and six healthy controls at rest and during 90 min cycle exercise, using the deuterium-labeled water method. All subjects served as their own control: on one occasion they were studied in the fasting state, and on the second occasion they received an infusion of triacylglycerol plus heparin. At rest, the fractional rate of gluconeogenesis from pyruvate was higher in patients than in controls in the fasting state. Triacylglycerol infusion was associated with increased rates of GNG(PYR) at rest in controls (p < 0.05) but not in patients. Circulating lactate and pyruvate levels were increased 3-fold during exercise in the CID patients. During exercise, GNG(PYR) increased in the CID patients (p < 0.01) and remained unchanged in controls, resulting in 85% and 72% higher absolute rates of GNG(PYR) in the patients than in the controls during fasting and triacylglycerol infusion, respectively. During exercise, rates of GNG(PYR) were not different between fasting and triacylglycerol infusion within both groups. Our data show that 1) GNG(PYR) is increased during exercise in CID patients; 2) increased pyruvate availability contributes to the higher rates of GNG(PYR) in the CID patients; and 3) exogenous infusion of fatty acids is not associated with increased rates of GNG(PYR) in CID patients at rest or during exercise. GNG(PYR) is a significant mechanism of lactate disposal in exercising CID patients, but triglyceride infusion does not enhance their lactate disposal through this mechanism.