To investigate the effects of basement membrane proteins on the reconstruction of mucosa equivalent, oral mucosa substitute were cultured on (1) type I collagen gels, (2) type IV collagen-coated type I collagen gels, (3) laminin-coated type I collagen gels, and (4) type I collagen gels containing both type IV collagen and laminin. H/E and PAS staining showed that the characteristics of the oral mucosa were preserved under all the experimental conditions. However, the basal keratinocytes appeared cuboidal when the type I collagen gels were coated with type IV collagen plus laminin. The expression of the differentiation markers was similar, but weak staining of filaggrin, K13, and involucrin was observed with the type IV collagen plus laminin coating. Furthermore, electron microscopy revealed that the size of the basal keratinocytes was relatively small and uniform when both type IV collagen and laminin were used. These findings suggested that these two major basement membrane proteins are important in the process of differentiation in mucosal keratinocytes.