Fish otoliths are known to be made of calcium carbonate and form daily rings, whose formation is controlled under circadian rhythm, and which have been used for daily age assessment. But, its detailed mechanism was unknown. We identified two matrix glycoproteins, one (OMP-1) from EDTA-soluble fraction and the other (otolin-1) from detergent-soluble fraction from chum salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss). It was found that OMP-1 and otolin-1 are loosely connected to each other in the otolith and are associated with daily ring formation. Immunohistochemical analysis clarified the producing cells of these glycoproteins only in the inner ear. Through knockdown experiments using zebrafish embryo, it was found that OMP-1 controls the growth of otoliths and otolin-1 maintains the positioning and hardness of otoliths. Two other matrix proteins, OMM-64 and carbonic anhydrase, involved in otolith calcification were also identified from the inner ear of the rainbow trout.