The basement membrane underlying epithelium of skin is generally believed to be of epithelial origin, but a mesenchymal contribution to the basement membrane has not been directly examined. The purpose of this study was to directly evaluate both epithelial and mesenchymal contributions to the basement membrane. Fetal bovine keratinocytes cultured on the surface of collagen gels in the absence of fibroblasts did not produce an ultrastructurally recognizable basement membrane; however, when these cells were cultured in the presence of dermal fibroblasts a basement membrane at the keratinocyte-fibroblast interface was produced after 1 week which was very similar in biochemical composition and ultrastructural appearance to dermal-epidermal basement membrane in human skin. When dual species cultures of bovine keratinocytes and human fibroblasts were analyzed by indirect immunofluorescent microscopy (IF)1 with human specific antibodies against basement membrane components, dermal fibroblasts were shown to synthesize and deposit type IV collagen, type VII collagen, and laminin in a linear manner into the basement membrane zone. Fetal bovine keratinocytes cultured in the presence or absence of fibroblasts synthesized and deposited type IV collagen, type VII collagen, laminin, K-laminin, kalinin, and basement membrane associated heparan sulfate proteoglycan (HSPG) into the underlying basement membrane zone. In organ culture, a subpopulation of fibroblasts initially migrating from human foreskin explants was found to stain strongly for types VII and IV collagen and laminin by IF whereas after subculture all cells showed a uniform low staining. Based on these observations we propose that differentiated fibroblasts exist adjacent to epithelial tissues in vivo which produce basement membrane components and assist in basement membrane assembly.