The interface between denuded dentin and regenerative periodontal tissue was investigated in a rat alveolar bone defect model using morphological and immunocytochemical approaches. The dentin surface was surgically exposed along the palatal roots of maxillary first molars. At 3 weeks post treatment, animals were perfused and treated regions from decalcified mandibles were embedded in Epon for ultrastructural studies or LR White for post-embedding immunogold labeling. Thin tissue sections were incubated with antibodies against noncollagenous matrix (osteopontin, bone sialoprotein, osteocalcin and fibronectin) and plasma (alpha2HS-glycoprotein and albumin) proteins. While in some cases, regenerative events took place directly on the denuded dentin surface, the interface between the denuded dentin and regenerating periodontal tissue was frequently characterized by the presence of an interfacial zone. This zone sometimes showed an electron-dense, cement line-like, planar accumulation of organic material immunoreactive for osteopontin and bone sialoprotein. Immunolabeling for osteocalcin and alpha2HS-glycoprotein was moderate and diffuse throughout the interfacial zone, whereas labeling with antibodies to albumin and fibronectin resulted in a weak reaction. It is concluded that accumulation of bone sialoprotein and osteopontin is a primary event during the formation of regenerative cementum onto denuded root surfaces.