Emdogain® is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain® enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain® (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for α-smooth muscle actin, a myofibroblastic marker. Morphometry and 3H-proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain® (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain® exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain®. Staining for α-smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain®. In conclusion, Emdogain® does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain® on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.