The application of autologous ex-vivo expanded cytotoxic lymphocytes to cancer patients may help to control minimal residual disease. However, the number of effector cells and the resulting antitumoral activity that can be generated in vitro are remarkably variable. Thus, we separately assessed the proliferative and cytotoxic potential of CD56+ CD3- natural killer (NK) and CD56+ CD3+ T-cells in relation to their expression of CD25, CD69, and CD16 in vitro. Two-week lymphocyte cultures from peripheral blood (n = 51) and from G-CSF-mobilized progenitor cell harvests (n = 11) were performed repeatedly from 14 women with breast cancer throughout conventional- and high-dose chemotherapy. A large proportion of CD25+ cells on day 7 of the culture predicted high expandability (r = 0.69, p < 0.00001), while elevated expression of CD69 predicted augmented cytotoxicity (r = 0.72; p = 0.00001) and low expandability (r = -0.69, p < 0.00001). CD25 and CD69 expression were inversely correlated (r = -0.8, p < 0.0001). CD16 expression was not suited to predict functional properties. Additionally, NK-cells were sorted by FACS according to CD25 versus CD69 expression. In a [3H]thymidine incorporation assay the CD25+ NK-cell fraction exhibited a higher proliferation rate than did the CD69+ fraction in all of three experiments. Together, our data suggest that CD69 is a useful marker for cytotoxic activity of NK cells, whereas proliferative potential is indicated by CD25 expression. These findings should help optimizing the ex-vivo generation of large numbers of cytotoxic effector cells for immunotherapy.