Cystathionine beta-synthase (CBS) catalyzes the first of two steps in the transsulfuration pathway that converts homocysteine to cysteine, a precursor of glutathione, a major intracellular antioxidant. Tumor necrosis factor-alpha (TNFalpha), which is known to enhance production of reactive oxygen species, increased CBS activity and glutathione levels in HepG2 cells. Western blot analysis revealed that the higher CBS activity correlated with cleavage of the enzyme to a truncated form. This cleavage was suppressed by inhibitors of superoxide production or by transfection with an expression vector for manganese superoxide dismutase. The commonly used proteasome inhibitors, MG132 and lactacystin but not N-acetyl-Leu-Leu-norleucinal, suppressed the TNFalpha-induced response. Targeted proteolysis of CBS was also observed in livers of mice injected with lipopolysaccharide, which is known to induce TNFalpha. Together, these data reveal a novel and previously unknown mechanism of regulation for homocysteine-linked glutathione homeostasis in cells challenged by oxidative stress.