The composition of the extracellular matrix produced by epithelial cells in contact with metabolically inert substrata was studied with antibodies specific for type VIII collagen. Type VIII collagen was present in the cell layer of cultured gingival epithelial cells, and at the epithelium-substratum interface of an explant culture model for junctional epithelium. Samples of the epithelial attachment apparatus (EAA) in vivo were collected by removing the junctional cells directly attached to the tooth (DAT cells) and the associated EAA matrix. The amount of material collected was sufficient for biochemical analysis of both intra- and extracellular components of the junctional cell-EAA complex. Immunological examination of the samples revealed that type VIII collagen was associated with epithelial cells forming the EAA in vivo. We suggest that this collagen type functions in the extracellular space as an attachment-promoting factor for gingival epithelial cells at the tooth surface.