Lactate has been shown to be an important oxidative fuel. We aimed to quantify the total lactate oxidation rate (R-ox) and its direct vs. indirect (glucose that is gluconeogenically derived from lactate and subsequently oxidized) components (mg.kg(-1).min(-1)) during rest and exercise in humans. We also investigated the effects of endurance training, exercise intensity, and blood lactate concentration ([lactate](b)) on direct and indirect lactate oxidation. Six untrained (UT) and six trained (T) men completed 60 min of constant load exercise at power outputs corresponding to their lactate threshold (LT). T subjects completed two additional 60-min sessions of constant load exercise at 10% below the LT workload (LT - 10%), one of which included a lactate clamp (LC; LT - 10% + LC). R-ox was higher at LT in T [22.7 +/- 2.9, 75% peak oxygen consumption ((V)O-2peak)] compared with UT (13.4 +/- 2.5, 68% (V)O-2peak, P < 0.05). Increasing [lactate] b (LT - 10% + LC, 67% (V)O-2peak) significantly increased lactate R-ox (27.9 +/- 3.0) compared with its corresponding LT - 10% control (15.9 +/- 2.2, P < 0.05). Direct and indirect R-ox increased significantly from rest to exercise, and their relative partitioning remained constant in all trials but differed between T and UT: direct oxidation comprised 75% of total lactate oxidation in UT and 90% in T, suggesting the presence of training-induced adaptations. Partitioning of total carbohydrate (CHO) use showed that subjects derived one-third of CHO energy from blood lactate, and exogenous lactate infusion increased lactate oxidation significantly, causing a glycogen-sparing effect in exercising muscle.