Effects of nivalenol (NIV), a trichothecene mycotoxin, on hepatic drug-metabolizing activity and aflatoxin B1 (AFB1) metabolism were investigated in male rats. In rats fed the diets containing 6-12 ppm NIV for 2 or 4 wk, decreases in initial feed uptake, terminal weight gain and organ weights were evident. An increase in cytochrome P-450 activity was observed in the hepatic microsomes, and Western blot analysis revealed a transient increase in P4502B1/2, together with a slight induction of P4501A2. The activity of cytosolic glutathione S-transferase (GST) enzymes was also enhanced in the rats, and Western blot analysis demonstrated an elevation of GST 1-2. The formation of aflatoxin B1-DNA adducts (AFB1-DNA) was increased in experiments using hepatic microsomal preparations from rats fed the NIV diet, whereas supplementation with cytosol prepared from NIV-treated rats reduced the microsomal potential for adduct formation. In in vivo experiments, the AFB1-DNA concentration in NIV-treated rats was lower than that in the controls. These results suggest that activities of cytochrome P-450 and GST enzymes were increased in rats fed NIV for several weeks. Alteration of these phase I and phase 2 enzyme levels resulted in the modulation of AFB1 adduction to DNA in vitro and in vivo.