An antibody to the C-terminus of the monocarboxylate transporter MCT1 was used to study the precise cellular and subcellular distribution of this transporter in rat heart. Postembedding immunogold procedures revealed that the labeling in the heart was restricted to cardiomyocytes and concentrated along the plasma membrane, including the transverse tubules. Gold particles occurred with highest densities in intercalated disks, where they avoided desmosomes and gap junctions. Labeling was also associated with plasmalemmal invaginations having ultrastructural features typical of caveolae. Internal membrane compartments were unlabeled. Quantitative analyses following postembedding labeling showed that the distribution of gold particles across the plasma membrane was nearly symmetrical, indicating that the C-terminus of the transporter is situated very close to the cell membrane. In preembedding immunogold experiments, the gold particles were localized at the external aspect of the plasma membrane, suggesting that the C-terminus is extracellular. From the present data, it can be concluded that even under basal conditions the majority of the MCT1 molecules in heart is present in the myocyte plasma membrane, implying that there is a constitutive functional expression of this transporter. It follows that the increased transmembrane flux of lactate during exercise or in pathological conditions such as ischemia must be a result of altered substrate gradients rather than of translocation of MCT1 molecules to the plasma membrane.