The present study is aimed to elucidate the changes in glutathione S-transferase (GST) activity and GST subunit components in primary cultured rat hepatocytes. Enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as cosubstrate. The activity decreased at 48 hr, and subsequently increased and returned to levels initially observed at 12 hr by 120 hr. Phenobarbital caused an induction of GST activity in culture at 72 and 168 hr. Immunocytochemical studies were performed using a peroxidase-anti-peroxidase technique with three polyclonal antibodies: anti-Ya, Yb1 and Yp. With anti-Ya, hepatocytes were persistently positive up to 144 hr in cell culture. With anti-Yb1, hepatocytes were positive at 24 hr, though positivity then gradually decreased. On the other hand, with anti-Yp, cells were almost negative at 48 hr and became obviously positive at 96 hr. Immunoelectron microscopy with anti-Yb1 using the avidin-biotin ferritin method revealed ferritin particles in the ribosomes on endoplasmic reticulum as well as in the free cytoplasmic space. In conclusion, the GST subunit components are in a state of dynamic change in cultured rat hepatocytes, and overall time-dependent increase in the total activity of the enzyme can be accounted for by increased expression of the Yp subunit. Finally, the intracellular localization of Yb1 subunit was clarified in the present report.