The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene.