The results presented here provide evidence that the presence of Fas protein in target cells is essential to permit cytotoxicity (resulting in apoptosis) mediated by cloned CD4+ Th1 cells. Using mitogen-activated B cells as targets, antigen-dependent lysis by CD4+ Th1 effectors was observed with MRL/MpJ+ but not with MRL/MpJ-lpr targets. The congenic MRL/MpJ-lpr strain is defective in Fas expression. Target cells from various lymphoid tissues of C3H.MRL-lpr mice were also resistant to the lectin-dependent cytotoxicity of Th1 effectors, whereas C3H/HeJ targets were sensitive. Moreover, a rapid DNA fragmentation prior to 51Cr release was induced only in C3H/HeJ targets. Thus, cytotoxicity induced by Th1 effectors correlates with target Fas expression. In contrast to Th1 effectors, CD8+ cytotoxic T lymphocytes (CTLs) killed C3H.MRL-lpr targets. When cytotoxicity was assayed in the presence of EGTA and MgCl2, which chelates extracellular Ca2+ [(Ca2+)ext], only C3H.MRL-lpr targets became resistant to CD8+ CTLs. This (Ca2+)ext-independent cytotoxicity of both Th1 and CD8+ effectors could be inhibited with unlabeled C3H/HeJ thymocytes or with a transfectoma carrying a murine Fas-human mu gene construct. In comparison, C3H.MRL-lpr thymocytes and the nontransfected parental cell line were poor inhibitors. Our study demonstrates that CD4+ Th1 cells and CD8+ CTLs differ in their (Ca2+)ext-dependent cytotoxicity but share a (Ca2+)ext-independent cytotoxicity that requires participation of Fas molecules for cytotoxic signal transduction leading to target apoptosis.