To study the contribution of putative perforin-independent mechanism in the antigen-specific target destruction by cytotoxic T lymphocytes, CD8+ CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-I--) embryonic stem cells into blastocysts of RAG-2 (-I-) mice. When tested on normal concanavalin A blasts, these perforindeficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas-mediated pathway in antigen-specific cytolysis.