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We examined the controversial notion of whether lactate is directly oxidized by subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria obtained from red and white rat skeletal muscle. Respiratory control ratios were normal in SS and IMF mitochondria. At all concentrations (0.18-10 mm), and in all mitochondria, pyruvate oxidation greatly exceeded lactate oxidation, by 31- to 186-fold. Pyruvate and lactate oxidation were inhibited by alpha-cyano-4-hydroxycinnamate, while lactate oxidation was inhibited by oxamate. Excess pyruvate (10 mm) inhibited the oxidation of palmitate (1.8 mm) as well as lactate (1.8 mm). In contrast, excess lactate (10 mm) failed to inhibit the oxidation of either palmitate (1.8 mm) or pyruvate (1.8 mm). The cell-permeant adenosine analogue, AICAR, increased pyruvate oxidation; in contrast, lactate oxidation was not altered. The monocarboxylate transporters MCT1 and 4 were present on SS mitochondria, but not on IMF mitochondria, whereas, MCT2, a high-affinity pyruvate transporter, was present in both SS and IMF mitochondria. The lactate dehydrogenase (LDH) activity associated with SS and IMF mitochondria was 200- to 240-fold lower than in whole muscle. Addition of LDH increased the rate of lactate oxidation, but not pyruvate oxidation, in a dose-dependent manner, such that lactate oxidation approached the rates of pyruvate oxidation. Collectively, these studies indicate that direct mitochondrial oxidation of lactate (i.e. an intracellular lactate shuttle) does not occur within the matrix in either IMF or SS mitochondria obtained from red or white rat skeletal muscle, because of the very limited quantity of LDH within mitochondria.