||Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer worldwide. Geographically, its incidence is unevenly distributed throughout the world and the current prognosis for this cancer is poor. Genetic alterations of tumor suppressor genes (TSGs) are central to tumor development. In the present study, the role of a known TSG was studied in esophageal cancer. p16INK4a, a G1-specific cell cycle regulatory gene located on chromosome 9p21, shows frequent loss of heterozygosity (LOH) in many human malignant tumors. It has been found to have a high alteration frequency including promoter hypermethylation, deletion, and mutation among the ESCC cell lines and primary tumors, which is suggestive that p16INK4a is playing a critical role in tumor growth through the p16INK4a/pRb pathway in ESCC. To elucidate the genetic status and role of p16INK4a in three ESCC cell lines, HKESC-2, T-Tn6/TSIS2V, and SLMT-1S9, a series of p16INK4a molecular analyses including polymerase chain reaction (PCR), methylation-specific PCR (MSP), reverse transcription-PCR (RT-PCR), Western blot, and transfection assays were performed. The p16INK4a gene was inactivated by promoter hypermethylation and deletion in all three cell lines. No p16INK4a RNA or protein expression was detected in the ESCC cell lines. HKESC-2 and T-Tn6/TSIS2V were transfected with a full-length p16INK4a cDNA. The growth of p16INK4a transfected cells was significantly reduced in both cell lines, illustrating the functional significance of the gene in cell cycle progression. These studies should help us better understand the key role of p16INK4a in ESCC development.