Purpose: This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC. Experimental Design: Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining. Results: Phospho-mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor α (ERαnuc; P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor β (ERβnuc) levels did not differ (P = 0.67). Patients with high ERαnuc and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ERαnuc and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ERβnuc tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR. Conclusions: ERα and ERβ were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression.