Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line31P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol. © 2001 Wiley-Liss, Inc.