The salinity preference of Japanese eel Anguilla japonica during silvering was validated from the viewpoints of physiological ecology by examining the pituitary prolactin (PRL) mRNA expression and the otolith strontium:calcium (Sr:Ca) ratios of the eels of different sexual maturity stages collected in an estuary of Taiwan. The PRL possessed a putative signal peptide of 24 amino acids and a mature peptide of 185 amino acids. RT-PCR and real-time quantitative PCR analyses indicated that the transcript levels of PRL mRNA significantly decreased from yellow to silver stage. On the contrary, the Sr:Ca ratios at the otolith edge of silver eels, which recorded their latest experience of salinity history, significantly increased. The increase in otolith Sr:Ca ratios and the decrease in PRL mRNA expression are consistent with the timing of migration from freshwater to seawater during silvering of the eel.KEY WORDS: Japanese eel · Salinity preference · Otolith · Sr:Ca ratio · Prolactin · mRNA · SilveringResale or republication not permitted without written consent of the publisher