Objective: To develop dentin matrix for clinical application in combination with periodontal surgery and investigate the responses of human periodontal ligament (HPDL) cells to dentin matrix compared with bone matrix in vitro. Since dentin matrix functions in facilitating follicular cells to differentiate into cementoblasts/osteoblasts during root formation, it might be one of the switches responsible for triggering the differentiation of human periodontal ligament cells during periodontal tissue regeneration. Methods: Matrices were prepared from bovine dentin and alveolar bone, respectively. Cells were treated with 0.01-10 μg/ml of both kinds of matrix for 24-48 hours. Cell proliferation was assayed by 3H-thymidine incorporation, collagen synthesis was assassed by SDS-PAGE and the expression of non-collagenous protein was analyzed by RT-PCR. Results: 3H-thymidine incorporation revealed that at 0.01-10 mg/ml of both kinds of matrix could trigger cell proliferation. Dentin matrix could also enhance collagen synthesis seen at day 10 and 15. However, RT-PCR analysis showed that expression of osteopontin, bone sialoprotein and osteocalcin also increased with period of treatment with both types of matrix whereas epidermal growth factor receptor decreased. Conclusion: Dentin matrix as well as bone matrix has an effect on driving PDL cells to a stage of more differentiation. However, dentin matrix could exert a more prominent effect on enhancing matrix synthesis. These findings imply that dentin matrix could be a kind of materials effective for helping periodontal tissue healing and regeneration. (This work was supported by Overseas Research Grant of the Asahi Glass Foundation and Chulalongkorn University)