EphA2 is a member of the Eph family of receptor tyrosine kinases, which interact with cell-bound ligands known as ephrins. EphA2 expression was investigated by immunohistochemistry with an anti-EphA2 monoclonal antibody in 80 patients with esophageal squamous cell carcinoma (ESCC) who had undergone surgery. EphA2 overexpression was positive in 40 of the 80 patients (50%). A significant correlation was observed between EphA2 expression and regional lymph node metastasis (p=0.023), number of lymph node metastases (p=0.011) and poor degree of tumor differentiation (p=0.004). The survival rates of EphA2-positive patients were poorer than those of EphA2-negative patients (p=0.014). The 5-year survival rate of patients without EphA2 overexpression was 68%, whereas that of patients with EphA2 overexpression was 29%. EphA2 expression was also investigated in 7 ESCC cell lines (TE-1, -2, -8, -13, -15, TT and TTn) and 1 immortalized human esophageal keratinocyte cell line (CHEK-1). Western blotting revealed different levels of EphA2 expression in the 8 cell lines. EphA2 was expressed at a high level in the ESCC cell lines compared to CHEK-1. EphA2 phosphorylation was demonstrated in all cell lines. Northern blot analysis showed that EphA2 mRNA expression in TE-1 was greater than that in the other ESCC cell lines. The observation of small gaps on Western blot analysis of the ESCC cell lines suggests that there may be a mechanism for EphA2 regulation at the point of translation. In conclusion, EphA2 overexpression appears to be related to poor degree of tumor differentiation and lymph node metastasis in ESCC. Consequently, patients with EphA2 overexpression have a poorer prognosis than those without. EphA2 is a potential target to prevent ESCC cells spreading into the lymphatic drainage.