Abstract The role of endogenous cholesterol in corticosteroid biosynthesis has been examined in the hamster adrenal. (A) In hamsters sacrificed at different times of the day the following have been determined: plasma corticosteroid and cholesterol levels, endogenous cholesterol content of the adrenal, the corticosteroid secretion pattern in the adrenal incubated for 90 min. and the adrenal cholesterol content after 90 min of incubation. (B) The capacity of the hamster adrenal to incorporate 3H 2O into digitonin precipitable sterols has been compared with that of the rat. The concentration of adrenal free cholesterol is significantly lower at 19:00 h than at 11:00 h. The in vitro secretion pattern of corticosteroids parallels that of plasma corticosteroids. both having a maximum around 18:00 h. Hamster adrenals can incorporate about nine-fold more 3H 2O into sterols than rat adrenals and five- to seven-fold more than rat liver. Hamster adrenals seem to be largely capable of synthesizing cholesterol from small molecules of substrate. Endogenous adrenal free cholesterol content may well be involved in regulating its own synthesis.