ABSTRACT The glutathione S transferases (GSTs, EC 2.5.1.18) of rat brain were purified to electrophoretic homogeneity and determined their anionic and cationic proteins using isoelectric focusing as eight and five, respectively, ranging the pI values from 5.1 to 9.8. The SDS-PAGE and substrate specificity analysis of rat brain GSTs on treatment with Phenobarbital (PB) ranging from 12 to 48mg the expression of GST subunits on long term and short term treatment was specific in the order of Yb, Yd and Yc. To observe the cell modification histology and immunohistochemistry studies were conducted on the sections of the rat brain. At the level of cell, the PB and other molecules caused damage to brain. Gliosis was predominant in the rat brain. The GST proteins can be used as markers at first stage by observing Yb subunit, later at second stage by observing Yd subunit and finally at third stage by observing the prolonged production of Yc subunit which is more useful to remove the hydroperoxides. Therefore the GSTs can function as markers at various levels in the presence of inducers in rat brain.