To examine the effects of ethanol (EtOH) on rat liver glutathione S-transferase (GST, E.C. 22.214.171.124), a key phase 2 biotransformation enzyme, an in vitro system consisting of rat hepatocytes co-cultured with rat epithelial cells derived from primitive biliary duct cells was used. Cells were either untreated or treated for 10 days with 0.1 or 0.4% EtOH. Cytosolic GST activities were assayed spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) as substrates. When cultures were exposed to 0.4% EtOH the total GST activity towards the substrate CDNB increased by 35%. However, the activity of GSTs towards DCNB did not increase significantly in the cells treated with this concentration of EtOH. Affinity chromatography followed by reversed phase HPLC was used to separate the various GST subunits. EtOH was found to affect the GST subunit pattern. Total GST proteins increased after exposure to 0.4% EtOH; this was largely due to a three-fold and 2.5-fold increase in the GST mu-class subunits 3 and 4 respectively. Since EtOH induces GST expression in liver parenchymal cells, particularly the mu-class GSTs, its addition as a solvent to cultured hepatocytes or its use in vivo may result in important changes in the metabolism and toxicity of the xenobiotics under study.