Osteonectin, gamma-carboxyglutamic acid-containing (Gla) protein, and dentin phosphoprotein were covalently attached to sepharose beads and inoculated in solutions at two different degrees of supersaturation with respect to both octacalcium phosphate (OCP) and hydroxyapatite. In both solutions, the inhibitory activity towards de novo formation of calcium phosphate that these proteins display when freely dissolved in solution was completely eliminated when they were immobilized on the sepharose at concentrations of up to 5 micrograms/mg wet beads. In the solution that was more highly supersaturated with respect to OCP, the immobilized dentin phosphoprotein, moreover, was found to induce de novo formation of OCP in proportion to the concentration of the protein immobilized. For example, at 10 micrograms/ml of the immobilized dentin phosphoprotein, the induction period was reduced more than 50%. However, in the solution considerably less supersaturated with respect to OCP, none of the immobilized proteins were capable of inducing OCP or apatite deposition. These findings suggest that the immobilized dentin phosphoprotein could work as a nucleating substrate for the OCP phase in solutions where calcium and phosphate concentrations are sufficiently higher than equilibrium saturation levels for the OCP phase.