A third isometallothionein was shown to be present during metallothionein synthesis, particularly following induction by zinc (Zn). It appeared to be closely related to metallothionein-II and the relative amount decreased with time, with only a minor peak after repeated cadmium (Cd) loading. On-line column switching by high-performance liquid chromatography (HPLC)-atomic absorption spectrophotometry (AAS) is effective in analysing and characterizing isometallothioneins.