The liver displays unique immunological properties including the ability to remove aberrant cells and pathogens and to induce peripheral immunotolerance. We have previously demonstrated that hepatocytes can cause cell death by a CD95 ligand–mediated mechanism. Here, we provide evidence that hepatocytes can kill other cells via a perforin-dependent pathway. Using cultured woodchuck hepatocytes and human liver cells as well as freshly isolated woodchuck, mouse, and human hepatocytes, we show that hepatocyte-mediated death of CD95-deficient target cells requires microtubule polymerization, a feature of the granule exocytosis–mediated cytotoxicity. Neutralizing anti-perforin antibodies and short-hairpin RNA directed against perforin messenger RNA confirmed the involvement of perforin in hepatocyte-mediated cell killing. Conclusion: This study shows that hepatocytes express biologically competent perforin capable of killing susceptible cells and emphasizes the role of hepatocytes as cytotoxic effectors. This also is the first demonstration of perforin in a non-lymphoid cell type. (HEPATOLOGY 2008.)