The quartered rat adrenal glands were incubated with acetate-1-14C in Krebs-Ringer-bicarbonate buffer. Analysis of the specific activity of free, ester sterol and corticosterone in the subcelhilar components of the incubated tissue revealed the mitochondrialfree sterol are the main source for corticosterone biosynthesis. The specific activity of corticosterone isolated from the incubated tissue was higher, irrespective of the incubation condition used, than that of mitochondrial free sterol. The results obtained in this experiment did not exclude the possibility of the existence of an alternative pathway which does not involve cholestrol in corticosteroidogenesis in rat adrenal. However, from the fact that close correlation was observed between the specific activity of corticosterone isolated from the incubated tissue and medium and that of mitochondrial free sterol in any incubation conditions employed in this experiment, that possibility seems to be least likely.