The specificity analysis of a CD3+, WT31+, CD8+ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2+ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2+ lines, autologous HLA-A2- melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca2(+)-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF alpha) and, to a lesser extent, for lymphotoxin (TNF beta). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)-gamma-specific mRNA. Antibodies to TNF alpha, TNF beta and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF alpha and to IFN gamma almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.