Extracellular matrix proteins are considered to play essential roles in controlling the nucleation, growth, and organization of hydroxyapatite crystals during enamel formation. The effects of amelogenin and the 32-kDa enamelin proteins on apatite nucleation were investigated by a steady-state gel diffusion device containing 10% gelatin gels loaded with 0, 0.75%, and 1.5% (w/w) native porcine amelogenins. It was found that the induction time for hydroxyapatite precipitation was strongly increased by the presence of amelogenins, suggesting an inhibitory effect of apatite nucleation. Addition of 18 micro g/mL of 32-kDa enamelin to 10% gelatin also caused inhibition of nucleation. Remarkably, addition of 18 and 80 micro g/mL of 32-kDa enamelin in gels containing 1.5% amelogenin accelerated the nucleation process in a dose-dependent manner. Our observations strongly suggest that the 32-kDa enamelin and amelogenins cooperate to promote nucleation of apatite crystals and propose a possible novel mechanism of mineral nucleation during enamel biomineralization.