The p53 tumor suppressor gene is thought to play a critical role in the control of cell growth and tumor development. The p53 is the most commonly mutated gene found in human tumors. To study the role of p53 in esophageal tumorigenesis and the possible mechanism(s) which lead to p53 inactivation, we examined esophageal cancers for p53 alterations. By a combination of Southern blot and PCR analyses, we found that 58% of esophageal cancers contained p53 allelic deletions, and most of these tumors revealed mutations in the remaining allele. p53 gene mutations were examined in fifteen tumor samples by single strand conformation polymorphism and DNA sequence analyses. Four nonsense mutations were found which resulted from either base substitution mutations or deletion-generated frameshift mutations. Seven missense mutations substituted a single amino acid in different exons of the p53 hot spot region (exons 5-8). One single base pair deletion resulted in a reading frameshift, while the remaining mutation was silent. p53 mRNA expression was examined in eight esophageal cancers and their accompanying normal tissues by Northern analysis. Three tumors expressed aberrant-sized p53 transcripts and one revealed total lack of p53 mRNA expression. No abnormal p53 mRNA was observed in any accompanying normal tissues. Aberrant-sized mRNA was found in one adenocarcinoma and its adjacent Barrett's dysplasia, suggesting that genetic alteration of p53 may have occurred before flank carcinoma, early in neoplastic progression. In addition, abnormal p53 mRNA expression in two tumors correlated with p53 intragenic deletion and/or allelic deletion. p53 intragenic deletions flanked by short direct repeats were found in two esophageal cancers. One was forty-five bp deletion, involving all RNA splice consensus sequences at the 3{dollar}\sp\prime{dollar} end of intron 7; Northern blot analysis revealed a total lack of p53 mRNA expression in this tumor. The other deletion removed 5 nucleotides within exon 8, resulting in a reading frameshift. Short direct repeats were found at the deletion breakpoints of the p53 gene in both tumors, with both deletions removing one repeat and the sequence between the two repeats. These results imply that a "slipped mispairing" mechanism occurring during DNA replication may have been involved in the generation of deletions in these esophageal cancers. We conclude that multiple mechanisms lead to p53 inactivation in human esophageal cancers, and our results strongly suggest that inactivation of the p53 gene is involved in esophageal cancer formation.