The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-(13)C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-(13)C] cysteine. The presence of [1-(13)C]hypotaurine and [1-(13)C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-(13)C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-(13)C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.