Objective: Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for treatment of B-cell malignancies. Natural killer (NK) cells, through the activating receptor FcγRIIIa (CD16), play a major role in rituximab-mediated ADCC. We have studied the in vitro effect of NK stimulators, such as interleukin-15 (IL-15) and CpG oligodeoxynucleotides A-Class (CpG ODN A), in the enhancement of rituximab-mediated ADCC against B-cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMC), purified NK cells, or NK-depleted PBMC from healthy donors, were activated with IL-15 or CpG ODN A, and cocultured with B-lymphoma cells in the presence of rituximab to evaluate the enhancement of the cytotoxicity. Results: The rituximab-mediated ADCC of IL-15-activated PBMC was twofold compared to unstimulated PBMC (73% ± 7% vs 37% ± 5% respectively, p < 0.001). Similarly, rituximab-mediated ADCC was enhanced when PBMC were activated with CpG ODN A as compared to CpG ODN control (61% ± 11% vs 36% ± 8%, respectively, p = 0.02). Nevertheless, the ADCC of purified NK cells was increased only with IL-15. NK-depleted PBMC activated with either IL-15 or CpG ODN A showed no ADCC, suggesting that NK are the major effector cells. Furthermore, IL-15 or CpG ODN A-activated PBMC, but not activated purified NK cells, secreted large amounts of interferon-γ in the presence of rituximab-coated lymphoma cells. Conclusions: IL-15 and CpG ODN A enhance rituximab-mediated ADCC against B-cell lymphoma. Under these conditions, NK cells seem to be the main effector cells mediating ADCC. These findings suggest that these agents could be used as adjuvants in combination with rituximab for patients with B-cell lymphoma. © 2008 ISEH - Society for Hematology and Stem Cells.