Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine-HCl (GuHCl) and 4 M GuHCl - 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS-PAGE with a relative mass approximately 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented approximately 0.5% of the total labelled proteins secreted. It comigrated on SDS-PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.