We have proposed that 11β-hydroxysteroid dehydrogenase is composed of structurally independent units with 11β-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11β-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11- reductase accompanied the purification. Homogeneity of 11β- dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 110- dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 ± 0.06 μM for corticosterone and 17.3 ± 2.24 μM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar. (Endocrinology123: 2390–2398, 1988)