Although enhanced epidermal growth factor receptor (EGFR) signaling has been connected with glottic cancerogenesis, the precise mechanisms of its activation still remain unclear. The aim of the present study was to examine EGFR on protein level, confronting cellular pattern of expression and EGFR gene amplification in glottic carcinomas. Tissue microarray technology was applied for uniformity of results. Biopsy specimens of patients with glottic squamous cell carcinoma and simple hyperplasia (control samples) were immunostained for EGFR. Immunohistochemical EGFR reaction was analyzed as membrane and cytoplasm positive and compared with the presence of gene amplification obtained by fluorescent in situ hybridization (FISH) analysis, obtained previously on a large group of patients. The cytoplasmic distribution of the EGFR staining appeared as a primary property of some squamous carcinoma cells; different from the membranous reaction, the reactions were mutually exclusive. Significantly higher scores of cytoplasmic EGFR staining were found in carcinomas with gene amplification when the cell reaction was examined in the basal and suprabasal layer. Our results suggest that EGFR expression in squamous cell carcinoma is different with regard to tumor cell position in carcinoma with ERGF gene amplification, which could be a new indicator of differently driven EGFR signaling in glottic cancer. Such results with cellular pattern distribution of EGFR protein are worthy of further research.