To enable a relevant interpretation of otolith strontium : calcium (Sr/Ca) variations in terms of habitat shifts of eels, the Sr/Ca-salinity relationship in eel otoliths was validated. Downstream and upstream migrations of young eels were reproduced in the laboratory by transferring groups of fish every 2 months between aquaria filled with water coming from the Dordogne river (salinity = 0), the upper Gironde estuary (salinity = 5), the lower Gironde estuary (salinity = 25) and the coast (salinity = 30), which represented the salinity gradient observed in the Gironde-Garonne-Dordogne watershed. Ontogenetic changes in otolith Sr/Ca were assessed in two groups of control fish that were kept in one of either two constant salinities (fresh water or seawater). X-ray electron microprobe (wavelength dispersive spectrometry, WDS) analyses of Sr/Ca ratios in the otoliths showed that the change of aquarium was recorded as a Sr/Ca increase (downstream migration) or a Sr/Ca decrease (upstream migration). No ontogenetic effect was detected in otoliths of control fish outside glass eel marks in either group of fish. The electron microprobe (WDS) analysis of the Sr/Ca life (transected in several otoliths of eels caught in the Gironde-Garonne-Dordogne watershed) showed that some of them were migrant eels that had experienced one major habitat shift during their continental life.