This study aimed to determine the effects of salinity on metal uptake and metallothionein (MT) mRNA levels in tilapia exposed to three metal ions. Male Oreochromis niloticus × O. aureus juveniles (hereafter, "tilapia") were exposed to various concentrations (100, 500, and 1 ppm) of metal ions (Cd(2+), Cu(2+) and Zn(2+)) in freshwater and water with two levels of salinity (10 and 20 ppt) for 7 days. Tests were then performed to investigate the effects of salinity on metal concentrations and MT mRNA induction in the test subjects' organs. Saline decreased cadmium (Cd) uptake and MT mRNA fold induction in various internal organs, but it did not enhance MT mRNA induction in the gills. Exposure to Cu(2+) caused greater copper (Cu) levels in the brains, intestines and livers, but Cu uptake in the intestines and kidneys occurred only at 10 ppm. MT mRNA induction caused by Cu(2+) was observed in various internal organs, but it occurred in the gills only at greater levels of salinity. Exposure at greater salinities also decreased zinc (Zn) uptake and MT mRNA induction in all organs except the gills. Although greater salinity decreased Cd and Zn uptake, the metal content in the water correlated with the MT mRNA levels in most of the organs, except for the intestines. In conclusion, metal accumulations in the livers and kidneys of tilapia correlated with MT mRNA levels. The levels of MT mRNA in the livers and kidneys of tilapia might therefore be used as biomarkers of exposure to Cd(2+), Cu(2+) and Zn(2+) in water of various salinities.