The fish otolith is a hard tissue consisting of calcium carbonate and organic matrices. The matrix proteins play important roles in otolith formation, but little is known about the nature of these proteins. In this study, matrix proteins were extracted from the otoliths of rainbow trout, Oncorhynchus mykiss, and chum salmon, Oncorhynchus keta. EDTA-soluble matrix proteins were separated by SDS-PAGE, revealing two major components in the otoliths of both species with apparent molecular masses of 55 and 43 kDa. N-terminal and some internal amino acid sequences of the 55-kDa otolith matrix protein were determined. A cDNA fragment encoding this protein of O. mykiss was amplified by reverse transcription PCR using two degenerate primers designed from the amino acid sequences. A cDNA encoding this protein was obtained by screening a saccular cDNA library using the amplified cDNA fragment as a probe. Nucleotide sequence analysis revealed that the cDNA clone has a sequence of 2.5 kb and the open reading frame encoding 344 amino acid residues. Northern blot analysis showed that mRNA of this protein is expressed specifically in the sacculus, and consistently during the day.