To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed. However, in our clinic, such a treatment did not induce additional activation of T cells. To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant. Cultures using isolated blood mononuclear cells were used as a control. When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures. However, when stimulation was performed with CD3 mAb, CD8bright+ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor beta (IL-2R beta). This is in contrast to the situation found in MNC cultures where all CD8bright+ cells expressed CD25 or IL-2R beta to a high extent at the end of culture. When rIL-2 or recombinant interferon gamma (rIFN gamma) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright+ cells were induced to express CD25 or IL-2R beta. These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures. In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright+ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy. The presented results suggest that in vivo simultaneous administration of rIFN gamma or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8+ T cells than CD3 mAb only.