Recently, much interest has focused on the role of HLA class II antigens in T cell-T cell interactions. We have studied the stimulatory capability in the primary mixed leukocyte reaction and the primed lymphocyte reaction of 11 alloactivated, HLA-DR- or -DP-reactive CD4-positive T-cell lines (Ta). From 70 to 90% of the Ta were HLA class II-positive as judged by the reactions with HLA class II-reactive monoclonal antibodies, and the Ta carried the DR allospecificities of the original T-cell donor when typed in the microcytotoxic test using DR-specific alloantisera. Neither irradiated nor nonirradiated Ta stimulated primed lymphocytes directed against the relevant HLA class II antigens on the Ta. Interferon-gamma, recombinant interleukin 1, phorbol myristate acetate, calcium ionophore, and adherent cells had no effect on the stimulatory capability of Ta. The ability of irradiated Ta to stimulate in the primary mixed leukocyte reaction (median counts per minute (cpm) 5.5 x 10(3] was significantly lower than that of peripheral blood mononuclear cells (cpm: 44.0 x 10(3]. The stimulation by Ta was almost only seen when the Ta were specifically directed against the class II antigens of the responder peripheral blood mononuclear cells (i.e., in combinations with "backstimulation") (median cpm: 21,000). In mixed leukocyte reaction combinations without backstimulation, significantly weaker reactions were seen (median cpm: 1,000). This observation may explain previous controversies concerning the stimulatory capacity of Ta. Recombinant interleukin 2 significantly enhanced the very low mixed leukocyte culture responses induced by class II-incompatible Ta in combinations without backstimulation but had no significant effect on cultures with Ta autologous to the responder peripheral blood mononuclear cells. Thus, allogeneic class II-positive Ta can induce interleukin 2 responsiveness but lack accessory cell function(s) necessary for the induction of interleukin 2 production in primed and unprimed T cells.