An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets,human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre-treatment of SCCHN targets with exogenous interferon-gamma (IFN-gamma). This inhibition was dose-dependent, reproducible and consistently observed with various SCCHN cell lines. SCCHN cells pre-treated with IFN-gamma had a significantly higher expression of intercellular adhesion molecule-I (ICAM-I) and major histocompatibility complex (MHC) class I antigens compared with untreated target cells. No differences in expression of the SCCHN-associated antigens on these targets or in the formation of NK-SCCHN conjugates were found, using flow cytometry. IFN-gamma-pre-treated SCCHN cells were less effective in competing with untreated targets in cold target inhibition assays and in inducing cytokine production from NK cells in co-incubation experiments. Protective effects of IFN-gamma on target cell sensitivity to lysis were blocked by pre-treatment of target cells with actinomycin-D or cycloheximide. The susceptibility of the target cells was restored by removal of MHC class I antigens from their surface by acid stripping before ADCC. Our results suggest that the decreased ADCC seen with SCCHN targets pre-treated with IFN-gamma is related to post-binding events, possibly altered signaling from targets to effector cells, and requires protein synthesis in the target cells.