We have conjugated the murine monoclonal antibody (528) against the human epidermal growth factor receptor (EGFR) to mammalian pancreatic ribonuclease (RNase) via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and 2-iminothiolene (2-IT). The conjugate showed dose-dependent cytotoxicity against EGFR-producing squamous cancer cells (A431, TE8, TE5, Ca9-22) and no detectable cytotoxicity against EGFR-deficient small-cell lung cancer cells (H69). The cytotoxicity of the conjugate was positively correlated with the EGFR numbers of each cell line. The addition of excess 528 antibody to the medium protected A431 cells from the conjugate cytotoxicity. This immunoconjugate might be useful for targeted treatment of squamous cell carcinomas hyperexpressing EGFR.