In order to establish an in vitro experimental model for studying periodontal regeneration after surgical treatment, we co-cultured human periodontal ligament (PDL) cells with slices of either mineralized or demineralized dentin. The specimens were processed for electron microscopy after 3, 7, and 28 days in culture. At 3 days, the PDL cells migrated up the vertical surface of both the mineralized and demineralized dentin slices and formed a three-dimensional, wedge-shaped area at the periphery of the slice. At 7 days, collagen fibrils with definite banding were observed in the extracellular space near the PDL cells. At 28 days, the wedge area appeared to be filled with a significant number of collagen fibrils. A layer of densely packed fibrils formed between the PDL cells and the vertical surface of the demineralized dentin slices, some of which interdigitated with exposed matrix fibrils in the dentin. This finding seems to resemble the initial stage of the formation of new connective tissue attachment observed in vivo. On the other hand, no collagen fibrils were detected at the interface of the PDL cells and the mineralized dentin slices.