The effect of administration of phenobarbital to rats on four different enzyme systems in the metabolic pathway from cholesterol to taurocholic acid, on the metabolism of androst‐4‐ene‐3,17‐dione, and on the oxidative demethylation of aminopyrine was studied in liver homogenates. In homogenates of liver from phenobarbital‐treated rats the extent of 7α‐hydroxylation of cholesterol and the conversion of cholest‐5‐ene‐3β,7α‐diol into 7α‐hydroxycholest‐4‐en‐3‐one were not significantly increased as compared to control rats. The 12α‐hydroxylation of 7α‐hydroxycholest‐4‐en‐3‐one was about 50% lower in phenobarbital‐treated rats than in control rats. The 7α‐hydroxylation of taurodeoxycholic acid was about twice as high in phenobarbital‐treated rats as in control rats. The conversion of androst‐4‐ene‐3,17‐dione into polar products and the formation of formaldehyde from aminopyrine were about 4 and 2.6 times higher, respectively, in phenobarbital‐treated rats than in control rats. The results indicate that the 7α‐hydroxylase catalyzing the conversion of cholesterol into cholest‐5‐ene‐3β,7α‐diol and the 12α‐hydroxylase catalyzing the conversion of 7α‐hydroxycholest‐4‐en‐3‐one into 7α,12α‐dihydroxycholest‐4‐en‐3‐one are different from the drug‐metabolizing enzyme systems. Copyright © 1968, Wiley Blackwell. All rights reserved