In patients with cancer, antibody‐dependent cellular cytotoxicity (ADCC) may be used as a laboratory test or for enhancing immunotherapy with murine monoclonal or chimeric mouse/human anti‐tumor antibodies (mMAbs or cMAbs, respectively). We have established an ADCC assay with IgG1 cMAb SF‐25, using human squamous‐cell carcinoma of the head and neck (SCCHN) cell lines as targets. By flow cytometry, all SCCHN cell lines tested expressed the antigen recognized by cMAb SF‐25. Trypsinization of the cell monolayers facilitated binding of cMAb SF‐25 to the antigen on the cell surface of SCCHN targets. Using the PCI‐50 SCCHN cell line as a target coated with this cMAb at the optimal concentration of 1.0 μg/ml, normal human peripheral blood mononuclear cells (PBMC, n = 28) were found to mediate ADCC at a mean level of 283 ± 42 (SEM) lytic units (LU20/107 effector cells). Non‐adherent monocyte‐depleted PBMC and natural killer (NK) cells purified by negative selection mediated significantly higher levels of ADCC than unseparated PBMC against SCCHN targets. NK cells, defined as CD3‐CD56' cells, could be effectively armed by cMAb SF‐25, as confirmed by flow cytometry and ADCC assays. IL2‐activated armed NK cells mediated higher levels of ADCC than non‐armed NK cells. Binding of cMAb SF‐25 to NK cells and their ADCC were enhanced by pre‐incubation with polyethylene glycol. Arming of NK cells with chimeric antibodies should be considered in developing novel strategies for treatment of human SCCHN, especially in the adjuvant setting. © 1995 Wiley‐Liss, Inc.