The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.