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.