Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.