Previous studies showed that Y-1 cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20 alpha-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high greater than normal greater than low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high greater than normal greater than low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20 alpha-dihydroprogesterone by cells showing normal or low C/P values, but not those with high values. It is concluded that the transport of cholesterol to the inner mitochondrial membrane is regulated by at least two processes, namely the supply of substrate (cholesterol), which can saturate this transport mechanism, and some mechanism(s) that is stimulated by cAMP and is capable of stimulating transport above maximal levels produced by high concentrations of cholesterol alone. The second mechanism requires the synthesis of a new protein(s).