Astrocytes possess at least two pathways for pyruvate and thus lactate formation involving precursors derived from mitochondria. The present results suggest that malic enzyme is the preferred route for this process. Although overall lactate release appeared to be independent of extracellular lactate concentration, the incorporation of mitochondrial precursors was decreased by starvation, which is known to deplete astrocyte glycogen stores. Using 1-[13C]glucose in the presence of 10 mM lactate led to label incorporation into extracellular lactate which increased from 3.4 +/- 0.4 to 6.5 +/- 0.5% during 4 and 6 h incubation periods, respectively. Lactate production from glycolysis proceeded virtually unaffected by the extracellular lactate concentration. The total amount of lactate in the medium decreased, however, demonstrating that lactate was used as a substrate.