The concerted view is that cytosolic pyruvate is transferred into mitochondria and after oxidative decarboxylation further metabolized in the tricarboxylic acid cycle. Recently this view has been challenged. Based on experimental evidence from rat skeletal muscle it has been concluded that mitochondria predominantly oxidize lactate in vivo and that this constitutes part of an 'intracellular lactate shuttle'. This view appears to be gaining acceptance in the scientific community and due to its conceptual importance, confirmation by independent experiments is required. We have repeated the experiments in mitochondria isolated from rat soleus muscle. Contrary to the previously published findings we cannot find any mitochondrial respiration with lactate. Analysis of lactate dehydrogenase (LDH) by spectrophotometry demonstrated that the activity in the mitochondrial fraction was only 0.7 % of total activity. However, even when external LDH was added to mitochondria, there were no signs of respiration with lactate. In the presence of conditions where lactate is converted to pyruvate (external additions of both LDH and NAD(+)), mitochondrial oxygen consumption increased. Furthermore, we provide theoretical evidence that direct mitochondrial lactate oxidation is energetically unlikely. Based on the present data we conclude that direct mitochondrial lactate oxidation does not occur in skeletal muscle. The presence of an 'intracellular lactate shuttle' can therefore be questioned.