11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11 beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11 beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11 beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11 beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11 beta-HSD 1 hom human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11 beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11 beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11 beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11 beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11 beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.