Slices of bovine adrenal cortex, bovine corpora lutea and rabbit testes were incubated with 20α-hydroxycholesterol-7α-3H or with cholesterol-7α-3H with and without the trophic hormones ACTH (adrenal cortex) or ICSH (corpora lutea or testes) added in vitro. In confirmation of previous findings, the trophic hormones increased the conversion of cholesterol-3H to cortisol-3H (adrenal cortex), progesterone-3H (corpora lutea) and testosterone-3H (testes). On the other hand, the trophic hormones produced a slight decrease in the conversion of 20α-hydroxycholesterol-3H to the same steroid hormones. In the case of the corpus luteum this decrease was less when incubation was continued for longer times, but under none of the conditions tested in any of the 3 tissues was the conversion of 20α-hydroxycholesterol-7α-3H to the steroid hormones increased in the presence of the trophic hormones. In the case of the testis similar results were observed 1 hr after a single intravenous injection of ICSH. These findings were most readily explained by the suggestion that, in vivo and in vitro, trophic hormones accelerate steroidogenesis by specifically stimulating the 20α-hydroxylation of cholesterol. (Endocrinology82: 559, 1968)