The aim of this study was to investigate the smoking habit influence on DNA methylation status in the promoters of the cancer related-genes MLH1, hTERT and TP53 in oral epithelial cells of healthy subjects.DNA methylation analysis was performed using methylation-sensitive restriction enzymes (MSRE) in oral epithelial cells from non-smokers, smokers and ex-smokers.The investigated CpG dinucleotides located at HhaI and HpaII sites in the MLH1 gene promoter were observed to be fully methylated in the majority of DNA samples from the smoker group and statistical differences were found between non-smokers and smokers and between smokers and ex-smokers (p0.05), with the fully methylated condition found to be a common event in healthy oral epithelial cells.We conclude that smoking may induce changes in DNA methylation status in cancer-related genes of oral epithelial cells and that the cessation of smoking is capable of reversing this process. Based on our data, we suggest that DNA methylation status of the hTERT and MLH1 gene promoters are promising markers for screening a set of smoking-related alterations in oral cells.