Cytokine-induced killer (CIK) cells have been used as immunologic effector cells in clinical trials with tumor patients. During CIK cell culture the fraction of CD3+/CD8+ and CD94+ cells increased significantly. In this study, we examined the ability of CD94-enriched CIK cells to destroy chronic myelogenous leukemia (K562) and pancreatic cancer (DAN-G) cell lines. In chromium release assays we could show, that the CD94-enriched cell population has significantly higher cytotoxicity against both tumor cell lines as compared to a CD94-depleted population and unmodified CIK cells. In addition, we determined the expression of different NKG2 receptors which partially associate with CD94 on CIK cells. CIK cells showed higher expression for NKG2D- homodimers in comparison to CD94/NKG2A- and CD94/NKG2C-heterodimers. Interestingly, we could enhance the cytotoxicity of CIK cells against the K562 cell line after antibody-directed stimulation of NKG2C and CD94. Antibody-labeled CIK cells showed higher cytotoxic activity than unmodified CIK cells and only CD94-labeled CIK cells. Altogether, the CD94-enriched population achieved the highest percentage of tumor cell lysis. These findings may be of relevance for further use of CD94-enriched cells in clinical trials.