The enzyme 20-hydroxysteriod dehydrogenase (20HSD) is a progesterone catabolizing enzyme which is highly expressed in mouse ovaries and adrenals. Whereas the functional significance of ovarian 20HSD for the induction of parturition has been defined, regulation and distribution of 20HSD in the adrenal gland has not been determined. We demonstrate that the expression of adrenal 20HSD is restricted to the X-zone, a transient zone between the adrenal cortex and the medulla of yet unknown function. Adrenal 20HSD activity in male mice peaks at three weeks of age and disappears thereafter while 20HSD enzyme activity is maintained in adrenals from nullipara female animals. Testosterone treatment of female mice induces rapid involution of the X-zone that is associated with the disappearance of the 20HSD positive cells. Conversely, reappearance of 20HSD expression and activity in male animals is evident after gonadectomy. Moreover, pregnancy, but not pseudopregnancy, is accompanied by X-zone regression and loss of 20HSD activity. Pregnancy induced X-zone regression and abolished 20HSD expression is partially restored in animals that were kept from nursing their pups. We found that in addition to its progesterone reducing activity 20HSD also functions as an 11-deoxycorticosterone (DOC) catabolizing enzyme. The unaltered growth kinetics of the X-zone in 20HSD knock out animals suggests that 20HSD is not required for the regulation of X-zone growth. However, 20HSD expression and enzymatic activity in all experimental paradigms is closely correlated with the presence of the X-zone these findings provide the basis for 20HSD as a reliable marker of the murine X-zone.