Introduction: Ex vivo expansion of monocyte-derived dendritic cells (mDCs) and subsequent coculture with autologous cytokine-induced killer (CIK) cells is an established system to create specific and non-specific anti-tumoral immunity. mDCs constitute the most frequently applied DC subset in clinical studies. One recently published approach to optimize the immunological functions of the DC/CIK cell system is the replacement of interleukin (IL)-4 by interferon (IFN)-alpha in the maturation process of the DCs. Materials and Methods: The expressions of relevant surface antigens of IL-4-DCs and IFN-alpha DCs by flow cytometry and the anti-tumoral activation of effector cells cocultured with both types of DCs using cytotoxicity assays were compared. In addition, short-term coculture experiments with both types of DCs and IFN-LAK effector cells were performed and compared with standard CIK cell coculture experiments. Results: Regarding the expressions of functionally relevant surface markers, no differences could be detected for CD80, CD83, and HLA-DR between IFN alpha-DCs and IL-4-DCs, whereas the mean fluorescence intensities of CD40, CD86, CD54, and HLA-ABC were decreased and the expression of CD14 was increased for IFN alpha-DCs. Moreover, no enhancement of cytotoxicity of cocultured CIK cells against tumor cell lines (A498 and SW480) was detected by the use of IFN-DCs. Additionally, coculture experiments with IFNgamma-LAK cells were performed and unexpectedly higher lysis rates in comparison with the established IL-4-DC/CIK coculture model was observed. Early incubation of the mDCs with several CpG-ODNs failed to increase the anti-tumoral cytotoxicity of the cocultured IFN-LAK cells. Conclusions: These results demonstrate that in the mDC/CIK cell system, IFN-DCs are not superior in inducing anti-tumoral cytotoxicity and even moderately inferior regarding the expression of functionally relevant surface markers compared with IL-4-DCs.