The interrelated signaling via TGF-beta1 and reactive oxygen species has a profound impact on fibrogenesis and is therefore selected as target for antifibrotic therapies. This prompted us to investigate the influence of the antioxidant N-acetyl-L-cysteine on TGF-beta signaling in culture-activated hepatic stellate cells, the most relevant pro-fibrogenic cell type in liver. Dissection of the molecular steps involved in TGF-beta signaling revealed that N-acetyl-L-cysteine dose-dependently abrogated the induction of the TGF-beta1 signaling reporter gene activation, the phosphorylation of Smad2 and Smad3, and the up-regulation of Smad7 mRNA. By means of Western blot analysis and cross-linking experiments, it was demonstrated that these effects are based on disintegration of TGF-beta1 and the TGF-beta receptor endoglin, as well as a reduced ligand binding capacity of betaglycan. We conclude that N-acetyl-L-cysteine is a specific inhibitor of TGF-beta signaling targeting different components of the TGF-beta signaling machinery. In conclusion, these findings suggest that this non-toxic aminothiol downregulates TGF-beta signal transduction thereby mediating beneficial effects on experimental liver fibrosis characterized by TGF-beta hyperactivity.