We have carried out an immunohistochemical investigation of xenografts of epidermal growth factor receptor (EGFR)-overexpressing tumors that have been induced to regress by treatment with rat monoclonal antibodies (mAbs) to the human EGFR [ICR16 (IgG2a), ICR62 (IgG2b), and ICR64 (IgG1)]. When mice bearing xenografts of the HN5 squamous cell carcinoma were treated for 5 days with mAb ICR62 or ICR16, the antibodies were found to be localized uniformly on the tumor cell membranes. However, the foci of tumor cells that remained following treatment with ICR62 were smaller than with ICR16 and the former showed a more pronounced host mononuclear cell infiltrate. Examination of the few tumors that had not regressed completely and were still present as static nodules 77 days following the final treatment with anti-EGFR mAbs revealed significant levels of therapeutic mAb in the nonviable areas of the tumors. The microscopic areas of apparently viable tumor cells that did not stain when only secondary antibody was used stained positive when the sections were treated first with an anti-EGFR antibody. This suggests that loss of the target antigen was not a significant factor and that these residual cells might be eradicated by further treatment with mAb. Furthermore, the finding of keratinized areas in the tumors undergoing regression suggested that the carcinoma cells had undergone terminal differentiation following exposure to antibody. This possibility was supported by the finding that treatment of HN5 cells in vitro with mAbs ICR16, ICR62, or ICR64 resulted in the accumulation of cells in the G0-G1 phases of the cell cycle and expression of the terminal differentiation markers involucrin and cytokeratin 10. We found no evidence of apoptosis in such cells. We conclude that antibodies which block the binding of EGF and transforming growth factor alpha to the EGFR can inhibit the growth of EGFR-overexpressing tumors by directing terminal differentiation and that a further therapeutic benefit may be obtained via immunological mechanisms with rat IgG2b mAbs such as ICR62.