Photochemical internalization (PCI) is a novel technology of macromolecular delivery. By PCI, endocytosed membrane-impermeable therapeutic drugs are photochemically released from entrapment in endo-lysosomal compartments to the cytosol of target cells. In the present report, we describe the in vitro proof-of-concept for PCI of cetuximab-saporin, an immunotoxin targeting EGFR-expressing cells. This immunotoxin consists of the chimeric murine-human IgG1 monoclonal antibody cetuximab (C225 or Erbitux) bound to the type I ribosome-inactivating protein toxin saporin by a biotin-streptavidin linkage. The photochemical treatment enhanced the cytotoxicity of the immunotoxin in a synergistic manner in three different EGFR-expressing carcinoma cell lines derived from different tumor tissues (colorectal, HCT-116; prostate, DU-145; and epidermis, A-431). Both cytotoxicity of cetuximab-saporin and epifluorescence of Alexa488-cetuximab were evaluated by competition with cetuximab demonstrating specific binding and uptake of cetuximab-saporin in EGFR positive cells. In the EGFR-negative uterine sarcoma cell line MES-SA, neither binding nor preferential accumulation of Alexa488-cetuximab was detected. In addition, PCI enhanced the cytotoxicity of cetuximab-saporin to the same extent as streptavidin-saporin in the MES-SA cells. In conclusion, PCI enhances selectivity of the cytotoxicity of the immunotoxin cetuximab-saporin in EGFR-expressing cells.