Stable human T cell hybridomas were produced by cell fusion between PHA-activated human peripheral blood lymphocytes and human acute lymphatic leukemia cells of a CEM cell line whose proliferation had been inhibited by treatment with an irreversible protein synthesis inhibitor (emetine) and an irreversible RNA synthesis inhibitor (actinomycin D). Two hybrid cell lines (E10, F8) thus produced expressed OKT3-reactive antigen, HLA-A1 and -B8 antigens derived from CEM cells, and HLA-A2 antigen derived from PBL, and they have 10 more chromosomes than CEM cells. Furthermore, these cell lines continuously secreted lymphotoxin over 3 mo. E10 was found to produce MIF in addition to lymphotoxin. Cloning of E10 and the relationship between the functions and surface phenotypes of E10 sublines were examined. Lymphotoxin-producing hybrids (E10-15 and E10-37) express more OKT8-reactive antigen than OKT4-reactive antigen, while lymphotoxin and MIF-producing hybrids (E10-25, E10-42, and E10-43) express more OKT4-reactive antigen than OKT8-reactive antigen.