Epidemiological studies have demonstrated a strong association between human esophageal cancer and exposure to N-nitroso carcinogens. Esophageal tumors can be induced in experimental animals, especially in rats, by many N-nitroso carcinogens. In the present study, rat esophageal tumors induced by methylbenzylnitrosamine (MBNA) and MBNA-transformed esophageal cell lines were analyzed for activated protooncogenes. DNAs from four Fisher 344 rat esophageal papillomas were examined for their ability to induce morphological transformation of NIH 3T3 cells. One of four esophageal tumors was positive in this assay. Southern blot analysis of this NIH 3T3 transformant revealed that the transforming gene was an activated Ha-ras gene. The activating mutation in the Ha-ras gene was identified by amplifying and then sequencing the first exon of this gene. A GC----AT transition at the second base in codon 12 of the Ha-ras gene was detected. The tumor DNAs from the transfection-negative samples were also amplified, and sequencing analysis of the first exon revealed a GC----AT transition in codon 12. In addition, 14 formalin-fixed and paraffin-embedded rat esophageal papillomas were shown to contain the same mutation in one of the alleles of the Ha-ras gene. In contrast, no point mutation was found in codons 12, 13, and 61 of the Ha-, Ki-, or N-ras genes in MBNA-transformed rat esophageal cell lines. The GC----AT transition detected in the esophageal tumors by DNA sequencing was confirmed by slot blot oligonucleotide hybridization of the polymerase chain reaction-amplified DNAs. The fact that mutated Ha-ras genes were detected in the esophageal papillomas suggests that activation of this gene occurred early in the process of neoplastic progression. The point mutation detected in the Ha-ras gene appears to result from a direct genotoxic effect of MBNA involving formation of the O6-methylguanine adduct. Taken together, these studies suggest that the activation of the Ha-ras gene plays an important role in the induction of esophageal neoplasia in the Fisher 344 rat by MBNA.