Guided tissue regeneration (GTR) is a concept that evolved from the development of membrane barrier techniques which allow the repopulation of periodontal wounds by desirable cells, resulting in a so-called new attachment apparatus. To understand the biological mechanisms involved in membrane barrier-led periodontal healing, the histological localization of macromolecules phenotypical of bone and cementum formation was investigated in regenerating human periodontal tissues harvested after healing by placing barriers on teeth untreatable except by extraction. Using immunolocalization techniques, frozen sections of soft tissues and hard tissues under GTR barriers were stained with antibodies to osteonectin (LB-BON-II) and bone sialoprotein (BSP) (LF-6); alkaline phosphatase (AP) was detected histochemically. Frozen sections of regenerating periodontal tissue demonstrated the presence of spindle-shaped, fibroblast-like cells entrapped in a dense fibrillar extracellular matrix. Rounded cells aggregated to form nodules heavily stained by the Alcian blue method, indicating the presence of proteoglycans and strongly resembling those noted in hard-tissue sections. At the electron-microscopic level, the cytoplasm of the elongated cells had numerous cisternae of endoplasmic reticulum and Golgi saccules, indicating metabolic activity. Striated collagen fibres were scattered throughout the field of the sections. AP-stained soft-tissue sections demonstrated the presence cell-bound and extracellular AP. Osteonectin antibody staining confirmed the presence of this macromolecule in the extracellular matrix, particularly in the area of the cellular nodules. The dense network of connective tissue fibres was also stained.(ABSTRACT TRUNCATED AT 250 WORDS)