A novel noncollagenous acidic protein was identified from the scales of goldfish (Carassius auratus), a freshwater teleost. Using an in vitro calcium phosphate crystallization assay, the EDTA-soluble fraction from these scales was screened for crystallization inhibitory activity, and a highly phosphorylated glycoprotein, named goldfish scale protein (GSP)-37, was isolated through 5 HPLC purification steps. The cDNA for GSP-37 has an open reading frame encoding a precursor protein, consisting of a signal peptide and GSP-37, with 19 and 137 amino acid residues, respectively. The C-terminal region of GSP-37 contains the RGD consensus sequence for cell adhesion. Although native GSP-37 strongly inhibited crystallization, alkaline phosphatase treatment dramatically reduced its inhibitory activity. Reverse transcription-PCR analysis revealed that GSP-37 is expressed only in scales but not in other calcified tissues, bones or pharyngeal teeth. In situ hybridization demonstrated that GSP-37-expressing cells were localized in the central regions of regenerating scales, where organic matrices were actively synthesized and were not stained with either alkaline phosphatase or tartrate-resistant acidic phosphatase, osteoblastic and osteoclastic cell markers, respectively. Immunohistochemical analyses showed that GSP-37 is localized in the uppermost region of the bony layer of the scale, which is thought to correspond to the enamel or enameloid layer of vertebrate teeth. All these data strongly indicate that GSP-37 is deeply associated with calcification in fish scales.