Transforming growth factor-alpha (TGF-alpha) stimulates cell proliferation through interaction with its receptor, the epidermal growth factor receptor (EGFR), by activating its tyrosine kinase activities. The simultaneous overexpression of TGF-alpha and EGFR by tumor cells is thought to trigger the autocrine growth pathway, leading to uncontrolled proliferation. To examine their roles in rat esophageal tumorigenesis induced by the chemical carcinogen N-nitrosomethylbenzylamine (NMBA), TGF-alpha, and EGFR expression was evaluated in normal rat esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical analyses. Compared with the levels in normal epithelium, the TGF-alpha and EGFR mRNA levels in esophageal papillomas were 3.6 and 1.9 times higher, respectively. In the preneoplastic epithelium, although a trend of increased TGF-alpha and EGFR mRNA levels was observed, collectively there were no significant differences between preneoplastic and normal samples by RT-PCR analysis. In situ hybridization and immunohistochemical staining showed increased levels of TGF-alpha and EGFR mRNA and protein products in papillomas and in pronounced hyperplastic and dysplastic lesions. TGF-alpha and EGFR expression correlated with each other and with the expression of proliferating cell nuclear antigen, a marker for cell proliferation. These results suggest that disregulation of TGF-alpha and EGFR expression may contribute to autonomous cell growth and may play an important role in rat esophageal tumorigenesis induced by NMBA.