AbstractCells within the periodontal ligament have the potential to regenerate a periodontal connective tissue attachment on pathologically exposed root surfaces as well as on several material surfaces including titanium. However, rather than a periodontal connective tissue attachment, a fibrous encapsulation or chronic inflammatory response has been reported at the material connective tissue interface for most dental materials. Cementum is the first tissue of the periodontal connective tissue attachment to develop and the secretion of enamel matrix related proteins on the newly mineralized dentin surface precedes and is thought to induce cementum formation. Enamel matrix‐related proteins may also function in the adult because the application of an acid extract of porcine enamel protein matrix (Emdogain®, EMD) on pathologically exposed root surfaces has been shown to result in cementum regeneration. Therefore, the objective of the present study was to determine whether the application of EMD to materials that do not normally support cementogenesis in vivo would alter the in vitro phenotype of periodontal ligament (PDL) cells including the synthesis of cementum‐associated extracellular matrix proteins. Primary PDL cells were established from 21‐day‐old Sprague‐Dawley rats, and were cultured on four materials commonly encountered in dental practice (gutta percha, calcium hydroxide, amalgam, and super EBA cement) with and without the application of EMD. After 7 or 14 days of culture, total‐DNA content, collagen synthesis, alkaline phosphatase activity, and the synthesis of a 42‐kDa cementum‐associated extracellular matrix protein were determined. PDL cells cultured on all materials had decreased total DNA content. The application of EMD further decreased total DNA content. PDL cells cultured on gutta percha and calcium hydroxide with the application of EMD had similar levels of collagen synthesis and alkaline phosphatase activity but also expressed a 42‐kDa cementum extracellular matrix‐associated protein when compared to the other groups. These results suggest that EMD can alter the phenotype of PDL cells when cultured on these dental materials. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 172–179, 2004