Extensive oral mucosa loss from a variety of conditions is associated with significant functional morbidity and mortality. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-β1(TGF-β1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms expressed different levels of TGF-β1. When NHOK reached confluency in serum free medium(KBM), in test medium containing 1.2 mM Ca++ KBM NHOK were allowed to form multi-layers and differentiate. The purpose of this study were to investigate the mRNA level of TGF-β1, FGF-2, and TIMP-1 by RT-PCR analysis and also to evaluate the expression of TGF-β1 and involucrin in keratinocytes at different times of the onset of differentiation. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. Cultured NHOK in preconfluency under KBM medium expressed a significantly higher level of TGF-β1 relative to those grown in multi-layer forms, while the level of TGF-β1 mRNA gradually reduced to its lowest level at 7 days of growing cells in test medium. Cultured NHOK in preconfluency of KBM medium expressed a lower level of FGF-2 and TIMP-1 relative to those grown in multi-layer forms, while the level of FGF-2 and TIMP-1 mRNA showed the highest level at 3 days at gradually reduced to its lowest level at 7 days of growing cells in test medium. As a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. It suggested that the expression of TGF-β1 mRNA be consistent with the expression of FGF-2 and TIMP-1 mRNA in NHOK grown in high calcium medium during the terminal differentiation. But differentiated NHOK expressing higher involucrin mRNA could show constant espression of TGF-β1, FGF-2 and TIMP-1.