BACKGROUND: High-protein diets have been shown to increase energy expenditure (EE). Objective: The objective was to study whether a high-protein, carbohydrate-free diet (H diet) increases gluconeogenesis and whether this can explain the increase in EE. DESIGN: Ten healthy men with a mean (+/-SEM) body mass index (in kg/m(2)) of 23.0 +/- 0.8 and age of 23 +/- 1 y received an isoenergetic H diet (H condition; 30%, 0%, and 70% of energy from protein, carbohydrate, and fat, respectively) or a normal-protein diet (N condition; 12%, 55%, and 33% of energy from protein, carbohydrate, and fat, respectively) for 1.5 d according to a randomized crossover design, whereas EE was measured in a respiration chamber. Endogenous glucose production (EGP) and fractional gluconeogenesis were measured via infusion of [6,6-(2)H(2)]glucose and ingestion of (2)H(2)O; absolute gluconeogenesis was calculated by multiplying fractional gluconeogenesis by EGP. Body glycogen stores were lowered at the start of the intervention with an exhaustive glycogen-lowering exercise test. RESULTS: EGP was lower in the H condition than in the N condition (181 +/- 9 compared with 226 +/- 9 g/d; P < 0.001), whereas fractional gluconeogenesis was higher (0.95 +/- 0.04 compared with 0.64 +/- 0.03; P < 0.001) and absolute gluconeogenesis tended to be higher (171 +/- 10 compared with 145 +/- 10 g/d; P = 0.06) in the H condition than in the N condition. EE (resting metabolic rate) was greater in the H condition than in the N condition (8.46 +/- 0.23 compared with 8.12 +/- 0.31 MJ/d; P < 0.05). The increase in EE was a function of the increase in gluconeogenesis (DeltaEE = 0.007 x Deltagluconeogenesis - 0.038; r = 0.70, R(2) = 0.49, P < 0.05). The contribution of Deltagluconeogenesis to DeltaEE was 42%; the energy cost of gluconeogenesis was 33% (95% CI: 16%, 50%). CONCLUSIONS: Forty-two percent of the increase in energy expenditure after the H diet was explained by the increase in gluconeogenesis. The cost of gluconeogenesis was 33% of the energy content of the produced glucose.