We have studied expression of p53, a tumor suppressor gene, by using both immunohistochemistry and in situ hybridization in 20 cases of squamous cell carcinoma of the esophagus. Immunohistochemical analysis was performed by using monoclonal antibody pAb1801. Immunoreactive p53 was observed in the nuclei of the tumor cells in 17 cases. We used 35S-labeled anti-sense single-stranded synthetic oligonucleotide probe ON102, which hybridized with DNA sequence near the 5' end of p53, for in situ hybridization. In all the cases of invasive squamous cell carcinoma studied, no significant accumulation of p53 hybridization signals was observed in carcinoma cells. This result indicates that overexpression of p53 observed by immunohistochemical staining is not due to an increase in the steady-state level of p53 mRNA in frank carcinoma cells. In six cases of morphologically normal esophageal mucosa distant from carcinoma, accumulation of hybridization signals was observed in basal and parabasal cells of the mucosa. The mucosa of these cases was negative for p53 immunoreactivity except for one case showing sporadic positivity. Accumulation of hybridization signals was observed in foci of squamous dysplasia not associated with invasion in three cases.