We examined the relationship between p53 mutation, murine double minute 2 (MDM2) gene amplification, and human papillomavirus (HPV) infection in 72 esophageal squamous cell carcinomas. We identified p53 mutations in 29 tumors (40.3%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. Amplification of the MDM2 gene was detected by Southern blot hybridization in 13 (18.1%) of 72 tumor tissues and in 4 (33.3%) of 12 cultured esophageal squamous cell lines. All four cell lines with MDM2 amplifications showed overexpression of the MDM2 mRNA in Northern blotting. We observed HPV infection in 15 (20.8%) of 72 tumor tissues by specific PCR amplification and Southern blot hybridization. In most tumors, amplification of the MDM2 gene or infection of HPV was not associated with p53 mutations, except in four cases with p53 mutation and MDM2 amplification, and three cases with p53 mutation and HPV infection. Since p53 mutations, MDM2 overexpression, and HPV infection are all considered to abrogate the normal function of p53 protein, each of these genetic changes may be equally important in tumorigenesis. In addition, we found that patients with MDM2 amplification exhibited a significantly shorter survival period (P = 0.0053).