Transforming growth factor-beta (TGF-beta) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-beta fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF beta decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-beta-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-beta-stimulated collagen production. These results suggest that GSH depletion induced by TGF-beta may mediate TGF-beta-stimulated collagen production. In addition, we showed that TGF-beta stimulated superoxide production and increased release of H2O2 from the cells, whereas GSH ester decreased basal and TGF-beta + glucose oxidase-stimulated H2O2 release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-beta-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-beta-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-beta-stimulated collagen production and that the effect of GSH depletion on TGF-beta-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.