The ex vivo expansion of human T-lymphocytes is of increasing importance in medical applications, especially for the adoptive immunotherapy of viral infection and malignant diseases of immunosuppressed patients. In these the rapies, the cells of the patient (dendritic cells or lymphocytes for example) are isolated from the blood and subsequently cultivated, expanded, and/or genetically modified er vivo [1,2], Afterwards, these expanded or modified cells are transplanted back into the patient (Fig. 1). For example, patients with leukaemia are very susceptible to otherwise harmless infections like cytomegalovirus (CMV) after chemo- and radiotherapy or bone marrow transplantation. The use of CMV specific cytotoxic T-lymphocytes can reduce the risk of such infections. These lymphocytes are cultivated together with antigen presenting cells, leading to the proliferation of specific T-lymphocytes which act against CMV. Peptides or proteins of the viral capsid can act as such antigens. For one medical application 2(.)10(9) specific cells are necessary. This amount of cells can not be isolated from donated blood. Usually there are > 100 specific T-lymphocytes in one donation. In some cases (e.g. CMV) up to 10(5) specific cells can be found, but there is still a demand for cell expansion before therapy. It is very difficult to produce such an amount of cells with conventional cultivation techniques like tissue culture flasks or culture bags. There is also a permanent risk of contamination with microorganisms. Therefore biotechnological methods are necessary which unable the cultivation of such an amount of cells under controlled conditions with a reproducible course of events. A controlled change of culture medium, the process control of pH, pO(2), and temperature, the easy sampling during cultivation and collection of cells after cultivation is of increasing importance. To produce clinical relevant numbers of specific T-lymphocytes. 70 tissue culture flasks (T75) would be needed. With regard to easy sampling during cultivation and isolation of the cells before transplantation, we endeavour to produce cells in high density culture systems. The production of a relevant number of cells is possible in one culture system with cell retention. There are different systems available, for example fed-batch processes or perfusion systems with cell retention by filtration, or continuously centrifugation. The aim of this investigation was to optimize the conditions for the selective activation and cultivation of human T-lymphocytes in tissue culture flasks starting with mononuclear cells (MNC). Afterwards a transfer of the optimized culture conditions to a high density culture system is presented.