Cytotoxic T cells (CTL) not only act as effector cells, but can also serve as antigen-presenting cells (APC) for other CTL due to their expression of major histocompatibility complex (MHC) class I molecules. In the present study we show that independently derived CTL lines (CTLL) with specificity for an L(d)-presented nonapeptide corresponding to amino acids 168-176 of the immediate-early 1 (IE1) protein of murine cytomegalovirus not only lyse syngeneic but also allogeneic target cells, if the peptide is present during the cytolytic assay. Whereas a short peptide pulse is sufficient to render syngeneic cells susceptible to lysis, continued presence of soluble peptide is mandatory for the lysis of allogeneic target cells. This indicates a difference in the mechanisms involved. Syngeneic BALB/c B cell blasts (K(d)D(d)L(d)) and mutant BALB/c-H-2dm2 B cell blasts lacking the restricting Ld molecules (K(d)D(d)0) were lysed to a similar extent in the absence of the IE1 nonapeptide, provided that the IE1-specific CTL had been pre-incubated with the peptide before the cytolytic assay. Since the mutant cells cannot present the IE1 peptide, their lysis indicates an MHC-unrestricted, peptide-independent mode of recognition by the CTLL. In addition, proliferation of the CTLL takes place after incubation with the cognate peptide, even in the absence of professional APC. These data indicate inter-CTL antigen self-presentation, resulting in activation of the lytic machinery leading to peptide-independent bystander lysis of allogeneic as well as syngeneic target cells.