Caspase-8, a member of the caspase family, plays an important role in apoptotic signal transduction in mammals. Here we report the identification and characterization of the caspase-8 (casp8) gene in the zebrafish Danio rerio. The zebrafish casp8 gene has a genomic organization similar to mammalian casp8 genes, consisting of 10 exons. By chromosome mapping, we found that casp8 maps on linkage group 6 (LG6), a zebrafish chromosome segment orthologous to the long arm of human Chr. 2, which carries CASP8. In contrast, the zebrafish casp10-like gene and the cflar gene separately localize on LG9 and LG11, respectively, and these genes form a cluster with CASP8 on the human chromosome. This chromosomal segregation is unique to fish but not other vertebrates. Furthermore, we examined the function of zebrafish Casp8 protein in mammalian cells, and showed that it has pro-apoptotic activity when overexpressed. In addition, this molecule was capable of transmitting apoptotic signals mediated through not only Fas but also the TNF receptor in mouse Casp8-deficient cells. Expression analysis showed that casp8 is maternally expressed, and transcripts continue to be present throughout embryogenesis and into larval stages. These results show that zebrafish casp8 has a structure and function similar to mammalian CASP8 orthologs, and our study suggests that the role of caspase-8 in the apoptotic signal pathway has been conserved over at least 450 million years of vertebrate evolution.