T cell blasts and lines passively acquire MHC molecules in vitro. To determine the role of these molecules in immunoregulatory reactions, we examined whether T cell lines grown on irradiated F1 spleen cells were able to supply allogeneic MHC antigens for the stimulation of T cell proliferation. Immunofluorescence analysis demonstrates that autoreactive T cell lines grown with irradiated F1 spleen cells acquire allogeneic class II molecules and subsequently lose the MHC molecules within 4 days of coculture with syngeneic cells. The proliferative response of (H-2k x H-2d)F1T cells stimulated by a T cell line grown on (H-2k x H-2d)F1 cells is inhibited by the addition of hybridoma-culture supernatants containing anti-IAd as well as anti-IEk antibodies. The proliferation of the F1 T cells to the T cell line grown on H-2k spleen cells is only affected by supernatants containing anti-IEk antibodies. To investigate the role of acquired class I MHC antigens, we examined their ability to serve as antigens for cytotoxic cells. Anti-H-2k cytotoxic T cells are generated when H-2b T cells are cultured with an H-2b-derived T cell line, only if the line has been grown on (H-2k x H-2b)F1 cells. An H-2b-derived T cell line exposed to (H-2k x H-2b)F1 cells can be lysed by anti-H-2k cytotoxic T cells from a primary MLR. Similarly, an H-2k anti-H-2b cytotoxic T cell clone will kill an H-2k-derived T cell clone grown on (H-2k x H-2b)F1 spleen cells. These results demonstrate that passively acquired class I molecules can stimulate the generation of cytotoxic T cells that lyse cells expressing the class I antigens and that passively acquired class I molecules expressed on T cells serve as the target for cytotoxic T cells.