[3H]Squalene 2,3(S);22(S),23-dioxide was prepared by incubation of [3H]mevalonate with S10 rat liver homogenate in the presence of 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase (EC 5.4.99.7). Aerobic incubation of [3H]squalene 2,3(S);22(S),23-dioxide with S10 rat liver homogenate affords 24(S),25-epoxycholesterol with an efficiency comparable to that of the formation of cholesterol from squalene 2,3(S)-oxide. The product 24(S),25-epoxy-[3H]cholesterol was identified by reduction to 25-hydroxyl[3H]cholesterol and by benzoylation to 24(S),-25-epoxy[3H]cholesterol benzoate. Both the 25-hydroxy[3H]cholesterol and 24 (S),25-epoxy[3H]cholesterol benzoate were isotopically diluted and recrystallized to constant specific activity. Incubation of either [3H]squalene 2,3(S);22(S),23-dioxide or 24(S),25-epoxy[3H]cholesterol with S10 rat liver homogenate failed to produce any detectable 25-hydroxy[3H]cholesterol.