Abstract The activity of purified 12α-hydroxylase from rabbit liver microsomes was modulated by including protein fractions from rabbit liver microsomes and cytosol into the system. The microsomal protein fraction stimulated 12α- hydroxylation two times. The cytosolic fraction inhibited the reaction markedly. The microsomal 12α-hydroxylase stimulatory activity was labile and the cytosolic 12α-hydroxylase inhibitory activity was stable to mild heat treatment. Addition of ATP and MgCl 2 or NaF had no effect on the activities of the two protein fractions. The activity of the microsomal stimulatory fraction decreased upon storage but could be reactivated by addition of reduced glutathione to the system.