Ligation of the externally expressed Fas (APO1/CD95) molecule will initiate programmed cell death (apoptosis), in many mammalian developing and adult cells. Fas-induced apoptosis has not been demonstrated with the cells of any non-mammalian vertebrate. We immunostained suspensions of splenocytes from adult Xenopus laevis, the South African clawed toad, with a polyclonal rabbit anti-human Fas antibody raised against the amino acid residues 321-335 of human Fas. The binding was specific, as it was dramatically reduced by preincubation of the antibody with the Fas peptide used to make it, but not with a Fas-ligand (FasL) peptide. The binding was enhanced after in vitro exposure of the splenocytes to phytahemagglutinin (PHA), a T cell mitogen and apoptogen in this species. Sections of developing Xenopus larval tissue were also immunostained with the polyclonal rabbit anti-human Fas antibody. Consistent binding of thymocytes and splenocytes was not observed until early metamorphosis in these immunological sites. A monoclonal mouse anti-human Fas antibody, previously used to stimulate apoptosis in mammalian cells, induced significant levels of apoptosis in adult Xenopus splenocytes and additionally, bound specifically to a splenocyte extract, as assayed by ELISA. Thus, a molecule on Xenopus splenocytes shares both structural and functional homologies with human Fas, indicating the evolutionary conservation within vertebrates of this means of initiating apoptosis.