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A simple method for the rapid expansion of human CD4+ T cells with both helper and killer functions was established. CD4+ T cells separated from peripheral blood mononuclear cells using immunomagnetic beads were stimulated with immobilised OKT-3 monoclonal antibody (mAb) plus recom-binant interleukin 2 (rIL-2) in 96 well culture plates. After 6 day-culture, the CD4+ T cells were restimulated by immobilised OKT-3 mAb for an additional 24 h, then inoculated into concentrated rotary-tissue culture bag and cultured for further 9 days. This procedure yielded a 3000-fold increase in cell number (about 3-5 x 109 per bag). Most of the cells (over 96%) continued to express CD4+ antigen and retained their capacity to produce IL-2. The activated CD4+ T cells showed marked cytotoxicity against Fc receptor positive tumour cells in the presence of OKT-3 mAb. Moreover, we succeeded in a specific targeting of the expanded CD4+ helper/killer T cells to c-erbB-2 positive tumour cells by means of anti-CD3 x anti-c-erbB-2 bispecific antibody. These results suggested that our established simple system will be available for the expansion of large number of CD4+ helper/killer T cells which may provide an efficient strategy for adoptive tumour immunotherapy. Recent work has demonstrated that introducing 'local help' at the tumour site is an important goal for the induction of anti-tumour activity in tumour-bearing hosts (Nishimura et been demonstrated that adoptive tumour immunotherapy using lymphokine-activated killer (LAK)5 cells and cytotoxic T lymphocytes was effective in animal and clinical systems However, adoptive immuno-therapy might give better results if helper T cells were transferred into the locality of the tumour together with killer cells. To develop this helper/killer therapy, it is necessary first to establish a large-scale culture system for human CD4+ T cells. It has been demonstrated that culture of peripheral blood mononuclear cells (PBMC) in the presence of interleukin 2 (IL-2) caused the predominant growth of CD8+ T cells (Gullberg et al., 1983; Taylor et al., 1985) and the selective in vitro growth of CD4+ T cells in the presence of IL-2 has been considered to be difficult. The different IL-2 responsiveness of CD8+ and CD4+ T cells was demonstrated to be derived from their differential expression of p75 IL-2 receptor (IL-2R) (Nakamura et al., 1991; Ohashi et al., 1989). Recently, however, we demonstrated that stimulation of FACStar-sorted CD4+ T cells with immobilised OKT-3 monoclonal antibody (mAb) induced p75 IL-2R expression and IL-2 responsiveness of CD4+ T …