The purpose of this study was to investigate the mineral induction capacity in vitro of polyanionic proteins covalently bound to a surface. Rat dentin gamma-carboxyglutamate-containing protein of the osteocalcin type (Gla-protein), proteoglycan (PG), and phosphoprotein (PP-H), as well as phosvitin (PhV) and bovine serum albumin (BSA), were covalently linked to agarose beads. There were incubated at 37 degrees C in solutions with a Ca/P molar ratio of 1.67, [Ca][P] molar products in the range 1.0-1.8 mM2, and an ionic strength of 0.165. The incubations were performed at constant pH and composition conditions; no spontaneous precipitation occurred under these conditions. Mineral formation, as monitored by scanning electron microscopy (SEM), was induced by all immobilized polyanions, including enzymatically dephosphorylated PP-H and PhV. No mineral was induced by BSA. The mineral inductive capacity of immobilized polyanionic proteins, as judged by the SEM after identical incubations, was found to differ between the different ligands. The mineral induced by PP-H and PG was shown by X-ray diffraction to be apatitic. It was concluded that, although polyanionic proteins in solution may inhibit mineral induction and growth, very minute quantities of such molecules, when immobilized on a surface, induce mineral at physiological concentrations of calcium and phosphate ions. The data presented may be taken to suggest that PP-H and PG, and perhaps other polyanions, may possibly be responsible for mineral nucleation in dentin and bone. The results, however, also point to the rather limited specificity in this type of reaction.