Each extract of liver, kidney, heart, lung, small intestine and brain of rat was applied to a diethylaminoethyl (DEAE)-cellulose column (10 mM Tris-HCl, pH 8.0), and glutathione S-transferase (GST) activity in the DEAE-cellulose-unbound and -bound fractions was assayed by using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The ratio of the bound to unbound GST activity differed considerably from tissue to tissue. In lung, kidney, liver and small intestine, the DEAE-cellulose-unbound fraction contained more GST activity than the DEAE-cellulose-bound fraction, but the opposite was the case in heart and brain. We examined GSTs in the DEAE-cellulose-bound fraction of rat liver in detail. Most GSTs in this fraction had YbYb subunits, and in addition, this fraction contained GSTs having YbYn subunits or YaYa subunits. The antiserum was raised against GST having YbYb subunits (peak II). The double immunodiffusion studies showed that the DEAE-cellulose-bound fraction of each tissue contained GST (s) antigenically similar to peak II. This was confirmed by the result that each GST activity was inhibited by anti-peak II serum on immunotitration.