Abstract Glutathione transferases (GST) are dimeric enzymes that take part in many detoxification processes. A previous report described the use of a glutathione-Sepharose affinity matrix for the purification of human liver GST. The method involved the use of 5 m M glutathione in a high pH buffer, and yields were nearly 100%. This method and adapted techniques have now been applied to rat liver GST. Selective GST elution can be obtained in several different ways: by stepwise change of the pH and/or glutathione concentration, and by linear gradient elution. Gel electrophoresis showed, however, that none of the fractions contained pure GST isoenzymes. Also, less than 50% of the total rat liver GST was eluted with 5 m M glutathione, in contrast to the results with human liver GST. A glutathione concentration of 30 m M is necessary for quantitative desorption of rat liver GST from a glutathione-Sepharose column.