The epidermal growth factor receptor (EGFR)-RAS-RAF-mitogen-activated protein kinase signaling cascade is an important pathway in cancer development and recent reports show that EGFR and its downstream signaling molecules are mutated in a number of cancers. We have analyzed 91 Japanese head and neck squamous cell carcinomas (HNSCC) and 12 HNSCC cell lines for mutations in EGFR, ErbB2, and K-ras. Exons encoding the hot-spot regions in the tyrosine kinase domain of both EGFR (exons 18, 19, and 21) and ErbB2 (exons 18-23), as well as exons 1 and 2 of K-ras were amplified by polymerase chain reaction and sequenced directly. EGFR expression was also analyzed in 65 HNSCC patients using immunohistochemistry. Only one silent mutation, C836T, was found in exon 21 of EGFR in the UT-SCC-16A cell line and its corresponding metastasic cell line UT-SCC-16B. No other mutation was found in EGFR, ErbB2, or K-ras. All tumors showed EGFR expression. In 21 (32%) tumors, EGFR was expressed weakly (+1). In 27 (42%) tumors it was expressed (+2) moderately, and in 17 (26%) tumors high expression (+3) was detected. Overexpression (+2, +3) was found in 44 tumors (68%). A worse tumor differentiation and a positive nodal stage were significantly associated with EGFR overexpression (P = 0.02, P = 0.032, respectively). Similar to patients from western ethnicity, mutations are absent or rare in Japanese HNSCC. Protein overexpression rather than mutation might be responsible for activation of the EGFR pathway in HNSCC.