For providing costimulatory signals, we utilized anti-CD28 monoclonal antibody (mAb) for the in vitro culture of tumour-draining lymph node (LN) cells. The proliferation of B16 melanoma-draining LN cells in the culture with anti-CD3 mAb was remarkably enhanced by the addition of anti-CD28 mAb. In culture with both anti-CD3 and anti-CD28 mAb, the B16-draining LN cells produced a higher level of interferon-gamma, but not interleukin-4, than with anti-CD3 mAb alone. The B16-draining LN cells efficiently expanded in the culture with both anti-CD3 and anti-CD28 mAb and subsequently with a low dose of IL-2 (anti-CD3 plus anti-CD28/IL-2). The expanded cells consisted predominantly of CD8+ T cells and showed a specific cytolytic activity, in a major histocompatibility complex (MHC) class I-restricted manner, even without in vitro restimulation. In addition, the adoptive transfer of the B16-draining LN cells, expanded in the culture protocol of anti-CD3 plus anti-CD28/IL-2, showed a significant anti-tumour effect against metastatic B16 melanoma in combination with IL-2. The cured mice thus acquired a specific protective immunity. Moreover, this protocol was also moderately effective against poorly immunogenic 3LL carcinoma. Overall, our results suggest the potential for another immunotherapeutic strategy based on 'the costimulatory theory' other than vaccination with B7-transfected tumour cells.