The cytosolic glutathione S-transferases (GSTs) are a family of phase II detoxifying isoenzymes that catalyze the interaction of the tripeptide thiol glutathione (GSH) with a wide variety of reactive and often toxic or carcinogenic electrophilic substrates. Pancreatic GSTs, however, have only been partially characterized. In this study, pancreatic cytosolic GSTs from male Fisher 344 rats were semipurified by affinity chromatography and then analyzed for isoenzyme content by chromatofocusing (fast protein liquid chromatography) and for sub-unit content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. In addition, polyclonal rabbit antisera were produced against homodimeric isoenzymes purified from rat liver and kidney, including the α class isoenzymes 1-1 and 2-2, the μ class isoenzyme 4-4, and the π class isoenzyme 7-7. These antisera were used in immunohistochemical (IHC) studies of the distribution of the pancreatic GSTs. A range of 0.5 1.6% of the total protein in rat pancreatic cytosol was found to be GST protein. The most abundant subunits present were the IT subunit 7 and μ subunits 3 and 4. Using modified methodology, smaller amounts of the a subunit 2 and the μ subunit 6 were detected, whereas very small amounts of the α subunits 1 and 8 were present. The IHC demonstrated that the GSTs were in large part limited to the duct system of the exocrinee pancreas, with positive staining of endothelial cells and stroma observed for the α and μ subunits. Isoenzymes containing the a subunit 2 were preferentially expressed in centroacinar cells and small ductules, whereas those containing the μ subunit 4 and the π subunit 7 were more prevalent within larger ductules and ducts. The lumens of the largest ducts also contained the two subunits 4 and 7. It is concluded that the acinar cells of the exocrine pancreas may lack the protection against electrophilic toxic and carcinogenic agents provided by the ductular system by GSTs.