Squamous cell carcinoma of the esophagus is a lethal cancer and carries a poor prognosis because of late diagnosis. Identification of molecular markers may aid early diagnosis. We assessed the expression of CDKN2A/RB1 in the esophageal mucosa and its association with the histology. Esophageal biopsies were collected from 38 patients with no esophageal lesion (group 1), from iodine-negative areas of 108 alcoholics/smokers (group 2), and from tumor and nontumor areas in 41 patients with squamous cell carcinoma (group 3). The histologic diagnosis was compared with immunoexpression of CDKN2A/RB1. In group 1, histology showed normal mucosa/mild esophagitis and no expression of CDKN2A/RB1. In groups 2 and 3, the diagnosis was: normal mucosa (38.4%), esophagitis (44.4%), dysplasia and carcinoma in situ (2.8%), and carcinoma (14.3%). The immunoexpression of CDKN2A/RB1 increased in a stepwise manner from the normal mucosa, to esophagitis, dysplasia/carcinoma in situ, and carcinoma (P<0.01). CDKN2A/RB1 was not expressed in the esophageal mucosa of patients without risk factors. p16/pRb expression increased in a stepwise manner, according to the severity of histologic lesions, in biopsies from patients exposed to risk factors or with carcinoma. Esophageal mucosa exposed to risk factors with the expression of those proteins may be at risk for malignant transformation.