Abstract Cycloheximide inhibits the action of luteinizing hormone (LH) and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) on the synthesis in vitro of progesterone in the corpus luteum of the rat. The results of the present work show that this synthesis depends on the presence of a protein with a rapid turnover rate which is formed by the action of LH (or cyclic AMP). This phase of mechanism of action of LH does not require molecular oxygen. On the other hand, molecular oxygen is indispensable for the subsequent stages, which lead to the synthesis of pregesterone. The regulating protein may act by allowing interaction between cholesterol and mitochondrial 20α-hydroxylase.