Contributions of carbohydrate and fat metabolism to the removal of a lactate (Lac-) load were quantified in inactive soleus (SL), plantaris (PL), and white gastrocnemius (WG) rat hindlimb muscle. Male Sprague-Dawley rats were perfused for 60 min with normal perfusate (NP, n = 8) or a high-lactate perfusate (LP, n = 8), simulating ionic conditions found in arterial blood and plasma after intense exercise: Lac- = 11.0 mM, K+ = 7.88 mM, and pH = 7.15. Metabolite fluxes across the hindlimb were calculated from blood flow and arteriovenous differences. In NP, Lac- was continuously released (2.9 +/- 0.2 mumol.min-1 x 100 g-1). However, in LP, a rapid and significant uptake of Lac- increased muscle Lac- fivefold to 39.6 +/- 1.1, 33.1 +/- 2.2, and 28.8 +/- 1.7 mumol/g dry wt in SL, PL, and WG, respectively. Glucose and O2 uptakes were similar during LP and NP perfusion. Glycerol release increased eightfold to 3.3 +/- 0.7 mumol.min-1 x 100 g-1 in response to LP. Muscle ATP, creatine phosphate, glycogen, glycolytic intermediate, and triacylglycerol concentrations did not change. However, muscle lactate-to-pyruvate ratios were elevated in all muscles of the LP group postperfusion, indicating changes in the mass action ratio at the pyruvate dehydrogenase reaction. In LP, of 80 mumol of Lac- taken up, 11% was accounted for by increased muscle Lac-, 12-24% was oxidized, and 5% may have been involved in glycerol release. The remaining Lac- may have been involved in metabolic cycling along the glyconeogenic-glycolytic pathway and/or in triacylglycerol-free fatty acid substrate cycling.