We report a study on neural metabolism of long-term three-dimensional cultures of rat embryonic brain cells in stirred vessels. Our experimental setup was optimized to keep viable aggregate cultures with neuronal maintenance for up to 44 days. Results show that aggregate size and shape could be hydrodynamically controlled depending on the impeller design, avoiding necrotic centers or significant losses in cell viability. Aggregates were composed mainly of neurons until day 16, whereas an effective growth of the glial population was observed after day 21. Cell metabolic status was evaluated by quantification of several metabolites in the culture medium; amino acid metabolism was used as a marker of metabolic interrelationships between neural cell types. Furthermore, (13)C-NMR spectroscopy was used on day 31 to explore specific metabolic pathways: incubation with [1-(13)C]glucose for 45 hr produced an increase in label incorporation in extracellular alanine, lactate, and glutamine, reflecting mainly astrocytic metabolism. The contribution of anaplerotic vs. oxidative pathways for glutamine synthesis was determined: a 92% reduction in the pyruvate carboxylase flux during the first 41 hr of incubation suggested a decrease in the need for replacing tricarboxylic acid cycle intermediates. We believe that our data corroborate the aggregating cultures as an attractive system to analyze brain cell metabolism being a valuable tool to model metabolic fluxes for in vitro brain diseases.