T lymphocyte recognition of cell-associated minor lymphocyte stimulation (Mls) superantigen was studied by simultaneous flow cytometric measurement of T cell free ionized intracellular calcium ([Ca2+]i) with the fluorescent probe indo-1 and T cell binding to antigen-presenting cells stained with a long-chained, membrane-fixed cyanine dye. Cloned T cell-B lymphocyte antigen-presenting cell conjugate formation and increased T cell [Ca2+]i were antigen specific and tightly linked in four Mls-reactive T cell clones. The T cell-antigen-presenting cell conjugates were extremely stable and, like T cell [Ca2+]i elevation, were maintained for more than 2 hr. Three ligand-receptor pairs, (i) T cell receptor/antigen+class II, (ii) CD4/class II, and (iii) LFA-1/ICAM-1, were obligatory participants in T cell recognition of cell-bound superantigen since monoclonal antibodies to any of them blocked formation of cell conjugates. Despite previous reports, Mls recognition occurred by the conventional T cell recognition pathway with easily detected phosphoinositide hydrolysis. B cell activation greatly enhances their recognition by Mls-specific T cells.