In vivo 31P nuclear magnetic resonance (NMR) spectroscopy provides unique opportunities to study the biochemistry of an organ within the intact animal in a totally non-invasive way. We have used in vivo and in vitro 31P NMR spectroscopy to study steady state changes in the major phosphorus-containing metabolites of the rat liver in control and chronically ethanol-treated rats. Chronic (4 month) ethanol treatment caused a statistically significant increase in the inorganic phosphate and phosphodiester resonances of rat liver in in vivo 31P NMR spectra relative to pair-fed control rats. Phosphomonoester and adenosine 5'-triphosphate resonances, as well as intracellular pH, were not appreciably altered. The effects of chronic ethanol treatment were particularly apparent in the response of the liver to a metabolic challenge of glycerol. Glycerol is phosphorylated almost exclusively in the liver and metabolized predominately via glycolysis and gluconeogenesis. Our in vivo 31P NMR results after administration of glycerol showed a significant increase in the phosphomonoester resonance in the liver of chronic ethanol-treated rats but not for their pair-fed controls. In vitro 31P NMR studies of perchloric acid extracts of liver showed that the increase was due to an accumulation of sn-glycerol 3-phosphate. This effect is due to the NAD(+)-dependent glycerol 3-phosphate dehydrogenase step being inhibited in the chronic ethanol-treated rats. This glycerol test may be useful in assessing the ability of the liver to rapidly regenerate NAD+ in situ and may be a more sensitive indicator of redox imbalance than steady state ratios of redox pairs (e.g., lactate/pyruvate).