The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) is a progesterone-catabolizing enzyme that is highly expressed in mouse ovaries and adrenals. Although the functional significance of ovarian 20α-HSD for the induction of parturition has been defined, regulation and distribution of 20α-HSD in the adrenal gland has not been determined. We demonstrate that the expression of adrenal 20α-HSD is restricted to the X-zone, a transient zone between the adrenal cortex and the medulla of yetunknownfunction. Adrenal 20α-HSD activity in male mice peaks at 3 wk of age and disappears thereafter, whereas 20α-HSD enzyme activity is maintained in adrenals from nulliparous female animals. Testosterone treatment of female mice induces rapid involution of the X-zone that is associated with the disappearance of the 20α-HSD-positive cells. Conversely, reappearance of 20α-HSD expression and activity in male animals is evident after gonadectomy. Moreover, pregnancy, but not pseudopregnancy, is accompanied by X-zone regression and loss of 20α-HSD activity. Pregnancyinduced X-zone regression and -abolished 20α-HSD expression is partially restored in animals that were kept from nursing their pups. We found that in addition to its progesteronereducing activity, 20α-HSD also functions as an 11-deoxycorticosterone-catabolizing enzyme. The unaltered growth kinetics of the X-zone in 20α-HSD knockout animals suggests that 20α-HSD is not required for the regulation of X-zone growth. However, 20α-HSD expression and enzymatic activity in all experimental paradigms is closely correlated with the presence of the X-zone. These findings provide the basis for 20α-HSD as a reliable marker of the murine X-zone.