The separation of metallothionein isoforms by Mono-Q column chromatography by fast protein liquid chromatography is described and compared to separation on a DEAE-Sephadex A-25 ion-exchange column. The separation of two isoforms of rainbow trout (Salmo gairdneri) metallothionein was possible by DEAE-Sephadex A-25 chromatography, while only one form of perch (Perca fluviatilis) metallothionein was obtained with this method. However, by applying FPLC on a Mono-Q column it was possible to separate two isoforms of perch metallothionein. When comparing the chromatographic behaviour of rainbow trout and perch metallothioneins it was found that the two isoforms of perch metallothionein were eluted closer together than the two rainbow trout metallothioneins. Both forms of perch and rainbow trout metallothioneins lacked aromatic amino acids and histidine, and had a high cystein content (30 mol%). The molecular weight was estimated to be 6000 for the apothioneins. The results from this study demonstrate the usefulness of FPLC to improve the resolution of metallothionein separations.