Background: Enamel matrix derivative placed in the backs of rats induces numerous eosinophilic round bodies (ERBs) and hard tissue. ERBs were analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and by database analysis. Subsequently, a seven-amino-acid sequence was identified, and an artificial oligopeptide was synthesized with its sequence. The present experiment was carried out to clarify whether or not the peptide induces hard tissue formation in vivo. Methods: Synthetic oligopeptide was injected into the backs of 15 rats. After injection, the tissues were excised at various times and prepared for light and immune-light microscopy. Results: A white lump was macroscopically observed in each rat back 14 days after injection of 7.5 and 15 mg/mL synthetic oligopeptide, and endochondral ossification and bone formation were microscopically observed in one rat back 14 days after injection of 15 mg/mL synthetic oligopeptide. Conclusions: The synthetic oligopeptide is pure, 1,118 dalton, and apparently low-toxicity. It seems to produce hard tissues, such as cartilage and bone tissue. © 2009, Japanese Stomatological Society. All rights reserved.