The lipoidal derivatives of [3H]pregnenolone, prepared biosynthetically, were converted, by incubation with a mitochondrial-microsomal fraction from adrenal cortical tissue, into lipoidal derivatives of 17-hydroxy-pregnenolone and of dehydroisoandrosterone, thus proving that these pregnenolone derivatives can serve as substrates for 17-hydroxylase and for the lyase enzyme that converts a C21-17-hydroxy-20-ketol into a C19-17-ketosteroid. Three synthetically prepared esters of pregnenolone, the oleate, the linoleate, and the arachidonate, were also hydroxylated at C-17 by a similar adrenal preparation. With the synthetic substrates, however, the corresponding esters of dehydroisoandrosterone were not formed.