Aim: 11β hydroxysteroid dehydrogenase (11β HSD) catalyzes the interconversion of glucocorticoids to inert metabolites in man and rodents and plays a crucial role in regulating corticosteroid hormone action. The physiological role and regulation of 11β HSD type 2 in the adrenal gland remains obscure. Therefore, the aim of the present study was to establish the pattern of 11β HSD type 2 expression in rat adrenal gland under conditions of testosterone withdrawal. Material and Methods: We performed immunohistochemical analyses of adrenal gland sections of ethane dimethanesulphonate (EDS)-treated adult rats. Results: In controls, strong positive 11β HSD type 2 signals were detected in the adrenal cortex cells, but not in the medulla. We observed the lowest 11β HSD type 2 expression intensity 7 days after initial treatment with ethane dimethanesulphonate (EDS) followed by progressive increase in the immunoreactivity toward days 14 and 21. Maximal staining intensity of 11β HSD type 2 in the adrenocorticocytes was found by day 35 after EDS treatment. Conclusions: By using the EDS model the present study provides new data about 11β HSD type 2 expression in the adrenal gland under conditions of testosterone withdrawal of adult rats. Our results elucidate further the functional significance of 11β HSD system in rat adrenal gland and the regulatory role of testosterone in its activity.