Tumor necrosis factor- (TNF-) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF- on thapsigargin (TG)-induced store-mediated Ca²⁺ entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF- slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF- significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF- abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF- was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF- inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-.