The mechanism by which adrenocorticotropic hormone stimulates rabbit adrenal tissue to produce cortisol was investigated by following the time course of the metabolism of pregnenolone-4-14C by homogenates of adrenal tissue from adrenocorticotropin-stimulated rabbits. Both on the basis of 14C content and total mass of products formed the sequence of intermediates between pregnenolone and cortisol appeared to be 17α-hydroxy pregnenolone, 17α-hydroxyprogesterone, and 11-deoxycortisol, although the data did not permit the definite exclusion of 17α,21-dihydroxypregnenolone as a possible intermediate between 17α-hydroxypregnenolone and 11-deoxycortisol. The rate of progesterone and corticosterone formation exceeded that of 17α-hydroxypregnenolone and cortisol, respectively. Results of incubations in which 1.0 μmole of unlabeled 17α-hydroxypregnenolone or 17α-hydroxy progesterone was included as traps were consistent with the metabolic pathway from pregnenolone to cortisol indicated by the time-course studies. © 1969, American Chemical Society. All rights reserved.