The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelia as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.