Lactate released into the surrounding salt solution as well as the cellular lactate content were measured in cerebral primary cultures of mouse astrocytes and of mouse neurons. Any newly produced lactate was immediately released as lactic acid into the extracellular compartment via a lactate/proton cotransport. The astrocytic release was about 2,000 nmol × mg−1 × hr−1; the neuronal release was about 300 nmol × mg−1 × hr−1. However, if election transport was blocked with dinitrophenol, the neuronal lactate release was as high as the astrocytic one under normal conditions. High glucose (30 mM) and K+ (60 mM) increased lactate release of astrocytes but not of neurons. In contrast it was found that insulin (1 μM) exposure mainly stimulated neuronal lactate release rather than glial release. Adenosine stimulated both neuronal and glial release. Neither intracellular lactate content nor concentration changed significantly in either cell type under any conditions tested. The pathophysiological implications of these measurements are discussed.