OBJECTIVE: To investigate the origin of myofibroblasts in oral submucous fibrosis. METHODS: The oral keratinocytes and fibroblasts were isolated and cultured. The expression of the alpha-smooth muscle actin in the fibroblasts was examined by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: No difference was found in expression of alpha-smooth muscle actin between the fibroblasts that were directly stimulated by arecoline and the control. The expression of alpha-smooth muscle actin in the keratinocyte and fibroblast-cocultured group was higher than in the control group, and higher in fibroblasts cocultured with keratinocytes preprocessed by arecoline than in fibroblasts cocultured with keratinocytes without preprocessed by arecoline. CONCLUSIONS: The differentiation of myofibroblasts from fibroblasts in oral submucous fibrosis might be induced by the interaction of arecoline and keratinocyte.