Evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. Such a process implies the presence of specific monocarboxylate transporters on both cell types. Expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was studied in enriched cultures of mouse cortical astrocytes or neurons. It was observed that, at both the mRNA and the protein levels, astrocytes strongly expressed MCT1 but had very little if any MCT2. By contrast, neurons had high amounts of MCT2 mRNA, although MCT1 mRNA was also detected. Double immunofluorescent labelings with appropriate markers confirmed the cell-specific preference in the expression of MCT1 and MCT2, but they revealed that a subset of neurons expresses low to moderate levels of MCT1. Parallel immunocytochemical stainings of cultured neurons with the presynaptic marker synaptophysin showed that MCT2 expression is correlated with synaptic development. Although MCT2 and synaptophysin were not colocalized, their distribution was similar, and they were often closely apposed, suggesting that MCT2 could be associated with postsynaptic terminals. Interaction between astrocytes and neurons, as occurring in layered cultures, did not modify the levels of MCT1 and MCT2 expression or their distribution and cell-specific preference under the conditions used. However, a close apposition between neurites and MCT1-expressing astrocytic processes was apparent and developed as cultures evolved. In addition to providing an extensive description of MCT distribution in cultured cells, our data underscore the potential of such preparations for future studies on the regulation of MCT expression.