The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was measured to be 32.1 ± 5.4 nmol/mg of protein. During a 24-h incubation in a minimal medium lacking amino acids and glucose, the content of glutathione in these cultures was reduced to 52% of the original content. On refeeding of glucose, glutamate, glycine, and cysteine, glutathione was resynthesized. A maximal content of glutathione was found 4 h after refeeding, exceeding the amount of glutathione of untreated cultures by 72%. Maximal glutathione synthesis was observed only if glutamate, cysteine, and glycine were present. If successively each one of these amino acids was made limiting for the synthesis of glutathione, half-maximal contents of glutathione were found at 0.2 mM glutamate, 20 µM cysteine, or 10 µM glycine. Replacement of glutamate or glycine by other amino acids revealed the potential of astroglial cells to convert glutamine, aspartate, asparagine, proline, and ornithine into glutamate, and serine into glycine. These results demonstrate that the concentration of intracellular glutathione can serve as an indicator for the presence of metabolic pathways of amino acids in cultured cells.