1. The isolation and partial purification of 11beta-hydroxy steroid dehydrogenase from rat and guinea-pig liver microsomes has been achieved by conventional methods. 2. The efficiency of different 11-oxygenated steroids as substrates has been examined. The relative efficiencies confirm in the main the stereochemical theory of the enzyme-coenzyme-substrate complex that was proposed earlier on the basis of studies in vivo. Delta(4)-3-Ketones and 5alpha-hydrogen steroids are readily metabolized by the enzyme. 5beta-Hydrogen steroids and Delta(4)-3-ketones with certain large alpha-substituents are metabolized to a limited extent or not at all. Halogen substitution in the 9alpha-position enhances the rate of reduction of 11-ketones but blocks the oxidation of the related 11beta-ols. 3. 9alpha-Fluorocortisol is a competitive inhibitor of the oxidation of cortisol, but 9alpha-fluorocortisone is reduced at five to ten times the initial velocity of cortisone. 4. 11beta-Hydroxy steroid dehydrogenase activity has been found in liver microsomes of rat, guinea pig, rabbit and calf. 5. Relative substrate efficiencies and K(m) values are similar in whole (debris-free) homogenates, washed microsomes and acetone-dried powders of washed microsomes. 6. A variety of conditions have been examined for the observation of 11beta-hydroxy steroid dehydrogenase activity. NADP(H) is an efficient and NAD(H) a very poor coenzyme for the reaction.