Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl 3 , 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl 3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-β£ (TNF-β£) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl 3 , TNF-β£ antiserum, and uridine. Thus, it was hypothesized that TNF-β£ from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-β£ messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca 2Ψ ] i and release of TNF-β£ caused by endo-toxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactos- amine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-β£ release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat. (HEPATOLOGY 1999;29:737-745.) Abbreviations: mRNA, messenger RNA; TNF-β£, tumor necrosis factor-β£; [Ca 2Ο© ] i , intracellular calcium; cDNA, complementary DNA; PCR, polymerase chain reaction; G 3 PDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide.