OBJECTIVE Ethanol consumption and pathological conditions such as cirrhosis lead to a reduction of hepatic glutathione. Hepatic methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, the major methylating agent, is regulated in vivo by glutathione levels. We have previously shown that nitric oxide inactivates methionine adenosyltransferase in vivo by S-nitrosylation. In this study, we aimed to investigate the regulation by glutathione of methionine adenosyltransferase S-nitrosylation in rat liver. METHODS Rat hepatocytes and whole animals were treated with buthionine sulfoximine, an inhibitor of glutathione synthesis, and methionine adenosyltransferase S-nitrosylation and activity were determined. RESULTS In hepatocytes, buthionine sulfoximine led to the S-nitrosylation and inactivation of methionine adenosyltransferase. Restoring glutathione levels in hepatocytes treated with buthionine sulfoximine, by the addition of glutathione monoethyl ester, a permeable derivative of glutathione, led to the denitrosylation and reactivation of methionine adenosyltransferase. In whole animals, buthionine sulfoximine led also to methionine adenosyltransferase S-nitrosylation and inactivation. S-Nitrosylation and inactivation of methionine adenosyltransferase induced by buthionine sulfoximine in whole animals was prevented by glutathione monoethyl ester. CONCLUSIONS These results indicate that in vivo hepatic methionine adenosyltransferase exists in two forms in equilibrium, nitrosylated (inactive) and denitrosylated (active), which are regulated by both the cellular levels of nitric oxide and glutathione.