Increased glutathione (GSH) level occurs early during liver regeneration and in many drug and/or radiation-resistant tumors. Whether GSH level is elevated in liver cancer is unknown. GSH levels and expression of GSH synthetic enzymes were measured in hepatocellular carcinoma (HCC) and normal liver. GSH levels doubled in HCC. The mRNA levels of g-glutamylcysteine synthetase heavy subunit (GCS-HS) and GSH synthetase (GS) doubled, whereas the expression of GCS light subunit was unchanged. Nuclear run-on assay showed that the rate of gene transcription doubled for both GCS-HS and GS. In HCC, there is increased binding to anti-oxidant response, AP-1 and NF-kB, three cis-acting elements in the 5'-flanking region of the human GCS-HS important for its transcriptional regulation. The role of GSH in cell growth was examined by using HepG2 cells. Cell GSH level was varied by treating cells with cystine (0 to 0.2 mM) with or without GSH ester or buthionine sulfoximine. Cell GSH level correlated directly with growth rate. Finally, preventing the increase in GSH after two-thirds partial hepatectomy blunted liver regeneration. Thus, GSH level is increased during liver growth as a result of up-regulation of GCS-HS and GS. This increase, in turn, facilitates growth.