We examined the genomic status of the CDKN2 gene including de novo methylation of 5' CpG islands in primary and metastatic tumor samples from 31 patients with esophageal squamous cell carcinoma. One somatic frame shift mutation (1 of 31; 3.2%) was identified by PCR-single strand conformational polymorphism analysis and DNA sequencing. Homozygous deletion and de novo methylation of the gene were confirmed in 5 (16%) and 6 (19%) of 31 patients, respectively. Homozygous deletion and de novo methylation were significantly associated with silencing of gene expression (P < 0.01). Aberrations of the CDKN2 gene were detected in tumors with lymph node metastasis and muscular invasion (12 of 22; 54%) and in none of stage I tumors (0 of 9.0%; P < 0.05). These results suggest that homozygous deletion and de novo methylation are predominant mechanisms of inactivation of the CDKN2 gene and may be associated with metastatic and invasive phenotypes of esophageal squamous cell carcinoma.