The role of Kupffer cells in CCl₄-induced fibrosis was investigated in vivo. Male Wistar rats were treated with phenobarbital and CCl₄ for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl₃) or were fed glycine, which inactivates Kupffer cells. After CCl₄alone, the fibrosis score was 3.0 ± 0.1 and collagen protein and mRNA expression were elevated, but GdCl₃ or glycine blunted these parameters. Glycine did not alter cytochromeP-450 2E1, making it unlikely that glycine affects CCl₄ metabolism. Treatment with GdCl₃or glycine prevented CCl₄-induced increases in transforming growth factor (TGF)-β1 protein levels and expression. CCl₄ treatment increased α-smooth muscle actin staining (score 3.0 ± 0.2), whereas treatment with GdCl₃ and glycine during CCl₄ exposure blocked this effect (1.2 ± 0.5); there was no staining with glycine treatment. These results support previous in vitro data and demonstrate that treatment of rats with the selective Kupffer cell toxicant GdCl₃ prevents stellate cell activation and the development of fibrosis.