Our objectives are to obtain, by various approaches, information about the regulation of those steroidogenic processes that occur in adrenal microsomes. We propose to study the regulation of microsomal enzymes catalyzing the synthesis of these adrenal steroids by several different approaches: 1) in vitro incubation techniques using microsomes from the bovine adrenal cortex. Kinetic experiments will be carried out using two steroidal substrates each labeled with a different isotope (3H, 14C or 35S). The time course of the incorporation of the isotopes into a common product can give information as to whether one or both substrates are obligatory intermediates. 2) To continue our investigations of the cytoplasmic factors which are known to modulate the microsomal enzyme, 21-hydroxylase. 3) To attempt to solubilize the adrenal microsomal 21-hydroxylase and to resolve it into its component parts. 4) To synthesize and study various chemical probes, specifically steroidal nitrones, which mimic progesterone by binding to a) the active site of 21-hydroxylase or other enzymes that use this steroid as a substrate or b) receptors that bind this hormone specifically. 5) To extend our studies to a search for factors that control the activity of other microsomal enzymes that are necessary for the biosynthesis of cortexolone.