Because the maintenance of glycemia is essential during prolonged exercise, we examined the effects of endurance training, exercise intensity, and plasma lactate concentration ([lactate]) on gluconeogenesis (GNG) and hepatic glycogenolysis (GLY) in fasted men exercising at, and just below, the lactate threshold (LT), where GNG precursor lactate availability is high. Twelve healthy men (6 untrained, 6 trained) completed 60 min of constant-load exercise at power outputs corresponding to their individual LT. Trained subjects completed two additional 60-min sessions of constant-load exercise: one at 10% below the LT workload (LT-10%), and the other with a lactate clamp (LT-10%+LC) to match the [lactate] of the LT trial. Flux rates were determined by primed continuous infusion of [6,6-(2)H(2)]glucose, [3-(13)C]lactate, and [(13)C]bicarbonate tracers during 90 min of rest and 60 min of cycling. Exercise at LT corresponded to 67.6 ± 1.3 and 74.8 ± 1.7% peak O(2) consumption in the untrained and trained subjects, respectively (P < 0.05). Relative exercise intensity was matched between the untrained group at LT and the trained group at LT-10%, and [lactate] during exercise was matched in the LT and LT-10%+LC trials via exogenous lactate infusion. Glucose kinetics (rate of appearance, rate of disposal, and metabolic clearance rate) were augmented with the lactate clamp. GNG was decreased in the trained subjects exercising at LT and LT-10% compared with the untrained subjects, but increasing [lactate] in the LT-10%+LC trial significantly increased GNG (4.4 ± 0.9 mg·kg(-1)·min(-1)) compared with its corresponding control (1.7 ± 0.4 mg·kg(-1)·min(-1), P < 0.05). Hepatic GLY was higher in the trained than untrained subjects, but not significantly different across conditions. We conclude that GNG plays an essential role in maintaining total glucose production during exercise in fasted men, regardless of training state. However, endurance training increases the ability to achieve a higher relative exercise intensity and absolute power output at the LT without a significant decrease in GNG. Furthermore, raising systemic precursor substrate availability increases GNG during exercise, but not at rest.