Dietary restriction (DR) reduced the metabolic activation of aflatoxin B1 (AFB1) in rats. This reduction may be attributed to the decrease of cytochrome P-450-mediated AFB1 epoxidation and/or increase in the detoxification of AFB1 catalyzed by hepatic glutathione S-transferase (GST) and other phase II detoxification enzymes. In this study the effect of DR on male rat liver cytosolic GST activity toward AFB1-8,9-epoxide was studied. The chemically-synthesized AFB1-8,9-epoxide was used as the substrate in this assay, and the formation of AFB1-GSH conjugate was analyzed by HPLC. Male Fischer 344 rats fed DR diets (60% of the food consumption of ad libitum (AL)-fed rats) showed a 2.4-fold increase in GST activity when AFB1-epoxide was used as the substrate. The results from the enzyme kinetic study showed that DR increased Vmax of the liver cytosolic GST but not the Km. Acute DR has little or no impact on GST activity when 1-chloro-2,4-dinitrobenzene and 2,4-dichloronitrobenzene were used as substrates. The mouse liver GST activity toward AFB1-epoxide was 3-fold greater than that of phenobarbital-induced rats, 4.5-fold greater than DR rats, and 14.7-fold greater than the GST activity of AL rats. This direct assay of liver GST activity using AFB1-epoxide as the substrate is useful for studying AFB1-induced biomarkers, such as AFB1-GSH conjugation and AFB1-DNA adducts.