Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol—the substrate for the first enzyme, namely P450_scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca ²⁺calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca ²⁺calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cylic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca ²⁺calmodulin possibly by redistributing cellular Ca²⁺. Ca ²⁺calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomoyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca ²⁺calmodulin ) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.