The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB(389)EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC(50)s of 0.4-50 pM. The epidermal growth factor receptor (EGFR) density of these cell lines was determined by immunofluorescence microscopy, flow cytometry, and radioligand binding. These assays correlated well with each other and demonstrated EGFR levels of 15,000-230,000/cell for 13 of 14 cell lines. The cell line U138MG, which lacked EGFR, was the only cell line insensitive to DAB(389)EGF. Linear regression analysis showed a good correlation between EGFR density and DAB(389)EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB(389)EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.