Cell-free extracts with high 14 alpha-hydroxylase activity were prepared from induced vegetative cell cultures of Mucor piriformis by grinding in potassium phosphate buffer (0.05 M, pH 8.0) containing glucose (0.25 M), KCl (1 mM), glutathione (1.0 mM) and glycerol (10%). Although the ideal pH for preparing the cell-free extract from vegetative cells was 8.0, the pH optimum of the hydroxylase was found to be 7.6. Microsomes (2.0 mg) prepared from the crude cell-free extract hydroxylated progesterone to 14 alpha-hydroxyprogesterone in approximately 60% yields in 30 min in the presence of NADPH and O2. Microsomes prepared from the uninduced cells did not contain any 14 alpha-hydroxylase activity. The hydroxylase activity was inhibited to a significant extent by CO and p-chloromercuribenzoate whereas moderate inhibition was noticed in the presence of SKF-525A, metyrapone and N-methylmaleimide indicating the possible involvement of the cytochrome P-450 system in the reaction. The membrane bound hydroxylase was solubilized using Triton X-100 and the solubilized fraction contained nearly 35% of the original hydroxylase activity.