The stonelike otoliths from the ears of fish consist of calcium carbonate crystallites embedded in an organic matrix framework. The organic matrix has long been known to play a pivotal role in the biomineralization of otoliths, and different methods have been used to conduct investigations on it. A new sensitive method for the in situ study of the regular variations in the organic matrix composition of serial small yellow croaker otoliths by Raman microspectroscopy and mapping is described. The major collagen bands were always observed around 1,272 cm(-1) (amide III) and 1600-1690 cm(-1) (amide I), and 1443 and 2800-3100 cm(-1) (bending and stretching modes of CH groups, respectively). Aromatic amino acids, such as phenylalanine and tyrosine, were identified at 1,003 cm(-1) and at 830 and 853 cm(-1). Tryptophan was assigned at 1,555 cm(-1), and it was firstly found in otoliths. A regular calcification process in otoliths was observed in Raman spectral mapping results. Corresponding changes were clearly seen in the concentrations of the organic matrix and aragonite (CaCO3) in otoliths.