Phospholipid vesicles containing the transmembrane protein H-2Kk spontaneously fuse to form planar membranes when incubated on treated glass surfaces. Pattern photobleaching of fluorescent lipid probes indicates that these planar membranes are continuous and that the lipids are as mobile as they are in conventional fluid bilayers or monolayers. H-2Kk molecules in these planar membranes are immobile. These membranes stimulate cytotoxic T lymphocytes when cultured with immune spleen cells. The response to H-2Kk in planar membranes is greatly enhanced by the addition of supernatant from concanavalin A-stimulated spleen cells, indicating that relatively little antigen processing or presentation by accessory cells occurs. Cytotoxic T cells induced by purified alloantigen are found to be as susceptible to antibody blockade as are effectors from conventional mixed lymphocyte culture, where the antibody is directed against a T-cell surface antigen reputed to strengthen target cell adhesion through an interaction independent of major histocompatibility antigens.