Adoptive transfer of large numbers of donor-derived T-lymphocytesmay offer a promising treatment of a variety of viral and malignant diseases. The key step in this approach is the ex vivo generation of sufficient quantities of these cells in a short time.We have investigated the influence of several important cultivation parameters on the proliferation of human T-lymphocytes to develop a large-scale fermentation process usingdifferent types of stirred bioreactors. Such systems offer manypotential advantages over the static culture systems commonlyused today.Peripheral blood mononuclear cells of healthy but CMV positive donors were stimulated with monoclonal antibodies (anti-CD3 and anti-CD28) and Interleukin-2. The influence of osmolality, Interleukin-2 concentration, pH, oxygen tension, feeding strategyand temperature on T-cell proliferation was investigated and theoptimised conditions were transferred to a novel stirred suspension bioreactor with an especially designed magnetic stirrbar to minimize the shear force (working volume 550 ml) and a standard stirred vessel (working volume 1000 ml).Preferable conditions for the cultivation of primary T-lymphocytes were an osmolality of 276-330 mOsmol kg(-1),an Interleukin-2 concentration of 100 U ml(-1), a pH rangeof 7.0 to 7.3, an oxygen tension of 5-50% and a temperature of 38.5 degrees C. After 238 h of cultivation 2.8 x 10(9) cells in the stirred vesseland 1.5 x 10(9) cells in the suspension bioreactor were obtained with a percentage of T-cells >94%. The specificity of the cells wasmaintained during cultivation as proven by IFN-gamma secretionafter exposure to a hCMV protein.