Aberrant promoter hypermethylation is common in oral squamous cell carcinoma (OSCC) and may be useful as a marker for cancer cells. The aim of our study was to assess whether the detection of hypermethylation of the promoter region of p16(INK4a) in oral cytological samples could be a marker in patients with risk of developing an OSCC. Hypermethylation was detected in 20% (29/145) of the cases analyzed. These findings provide further evidence that the inactivation of p16(INK4a), due to methylation, is an early event in the carcinogenic pathway of OSCC.