Hexose-6-phosphate dehydrogenase was purified from rat liver microsomal fraction more than 500-fold with a 45% recovery using DEAE-cellulose and 2',5'-ADP-Sepharose 4B columns. The purified enzyme appeared to be immunologically and electrophoretically homogeneous and had broad substrate and cofactor specificities. The enzyme activity was not inhibited by p-chloromercuribenzoate. The purified enzyme was a glycoprotein in nature, having a Stokes radius of about 55 A, a sedimentation coefficient of about 8.2 s, and an isoelectric point of about 6.4. Minimum molecular weight of the enzyme was about 108,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the product cross-linked with glutaraldehyde or dimethyl suberimidate had Mr approximately equal to 220,000, suggesting that the active enzyme existed as a dimer of identical subunits. Antiserum raised against the purified enzyme inhibited the activity of the solubilized enzyme but did not inhibit the cytosol glucose-6-phosphate dehydrogenase activity. The antigenic sites of the enzyme were latent in intact microsomes. Comparison was also made between the enzymes isolated from untreated and phenobarbital-pretreated animals.